Optical analyte detection systems and methods of use

ABSTRACT

Various embodiments are drawn to systems and methods for detecting an analyte of interest in a sample including an optical sensor, a capture probe attached to a surface of the optical sensor wherein the capture probe is capable of binding to the analyte to form a duplex or complex, and an antibody capable of binding to the analyte, duplex, or complex. In several embodiments, systems and methods further include a particle attached to the antibody or capable of binding to the antibody. In several embodiments, systems and methods for analyte detection feature one or more of the following: high detection sensitivity and specificity, scalability and multiplex capacity, ability to analyze large analytes, and ability to detect or measure multiple individual binding events in real-time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/883,280, filed May 2, 2013, and entitled “OPTICAL ANALYTE DETECTIONSYSTEMS AND METHODS OF USE,” which is a national phase ofPCT/US2011/059454, filed Nov. 4, 2011, and entitled “OPTICAL ANALYTEDETECTION SYSTEMS AND METHODS OF USE.” This application claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/410,595 filed Nov. 5, 2010 and U.S. Provisional Application No.61/452,796 filed Mar. 15, 2011. All of the foregoing applications arehereby incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with United States Government support underGrant No. 1-DP2-0D002190-awarded by the National Institutes of Health(NIH) Director's New Innovator Award Program. The United StatesGovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledGNLYT002WO.TXT, created Nov. 3, 2011, which is 2.77 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD

Various embodiments provided herein are applicable to the fields ofoptics and analyte detection.

BACKGROUND

The ability to perform multiple simultaneous biomarker measurements incomplex samples with high sensitivity presents a large challenge todisease diagnostics and biological studies. Technologies such aspolymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), andcDNA microarrays have been used for comparative and quantitative globalDNA and mRNA expression studies. Two-dimensional polyacrylamide gelelectrophoresis (2D-PAGE) and immunoassays, such as the enzyme-linkedimmunosorbent assay (ELISA), have been used to analyze proteincomponents from complex mixtures. However, these technologies haveseveral limitations and suffer from low dynamic range, low sensitivity,low specificity, labor intensiveness, lack of scalability or multiplexcapability, inability to analyze large analytes, and/or inability todetect binding events in real-time. Moreover, many existing technologyplatforms, such as microarrays, are equilibrium based detectionapplications that are incapable of real-time binding detection, which isimportant for eliminating signal bias of non-specific binding. Anotherdetection platform, Surface Plasmon Resonance (SPR) sensors, has beenused to measure binding-induced changes in the local refractive index ofthe sensors, but is not amenable to large scale multiplexing oroperation in complex media or clinical samples. These drawbacks havelimited the widespread applicability of current detection platforms indiverse analytical settings.

SUMMARY

Various embodiments are drawn to systems and methods for analytedetection featuring one or more of the following: high dynamic range,high detection sensitivity and specificity, scalability and multiplexcapacity, ability to analyze large analytes, and ability to detect ormeasure multiple binding events in real-time with reduced cross-talkfrom non-specific binding events. Furthermore, the systems and methodsof various embodiments may involve low sample volume in the microliterrange, only a relatively small amount of hands-on time, and providerapid time to results, which are reproducible. Unlike existing analytedetection platforms, the systems of various embodiments are not impairedin wide-range applicability by having capacity for only some beneficialdetection properties at the expense of others. Various embodiments ofthe systems and methods provided herein can potentially overcome thetechnical drawbacks that have hampered current detection platforms frombeing useful across a wide spectrum of contexts.

Several embodiments are drawn to a system for detecting a nucleic acidmolecule of interest in a sample including an optical sensor; a nucleicacid capture probe attached to a surface of the optical sensor, whereinthe capture probe is capable of hybridizing to the nucleic acid moleculeof interest to form a duplex; and an antibody capable of specificallybinding to the duplex of the capture probe and nucleic acid molecule ofinterest, wherein said optical sensor has an optical property that isaltered when said antibody is bound to said duplex such that saidoptical sensor is configured to sense said antibody combined with saidduplex.

Various embodiments relate to a system for detecting a nucleic acidmolecule of interest in a sample including an optical sensor configuredto resonate at a resonant wavelength; a light source capable ofproviding light at said resonant wavelength for the optical sensor; anucleic acid capture probe attached to a surface of the optical sensor,wherein the capture probe is capable of hybridizing to the nucleic acidmolecule of interest to form a duplex; an antibody capable ofspecifically binding to the duplex of the capture probe and nucleic acidmolecule of interest; and a detector, wherein said optical sensor has anoptical property that is altered when said antibody is bound to saidduplex such that said optical sensor is configured to sense saidantibody combined with said duplex and the detector is capable ofdetecting the optical property that is altered. The light source maycomprise in various embodiments a laser such as a tuneable laser orbroad band light source such as a superluminescent laser diode (SLED).

Some embodiments are directed to a system for detecting an analyte ofinterest in a sample including an optical ring resonator, a captureprobe attached to a surface of the optical ring resonator, wherein thecapture probe is capable of binding to the analyte to form a complex,and an antibody capable of binding to the analyte or complex, whereinsaid optical ring resonator has an optical property that is altered bysaid antibody bound to said complex or analyte, when the analyte isbound to the capture probe, such that said optical ring resonator isconfigured to sense said antibody combined with said analyte or complex.

In several aspects of embodiments provided herein, the nucleic acidmolecule of interest comprises deoxyribonucleic acid (DNA) orribonucleic acid (RNA). In further aspects, the capture probe comprisesa DNA oligonucleotide. In some aspects, the DNA oligonucleotide iscomplementary to the nucleic acid of interest or analyte of interest. Insome aspects, the DNA oligonucleotide comprises a modified DNAnucleotide, such as a locked nucleic acid (LNA) or universal base. Insome aspects of the aforementioned embodiments, the capture probecomprises an RNA oligonucleotide. In various aspects, the RNAoligonucleotide is complementary to the nucleic acid of interest oranalyte of interest. In several aspects, the RNA oligonucleotidecomprises a modified RNA nucleotide, such as a locked nucleic acid (LNA)or universal base.

In various aspects of embodiments provided herein, the antibody binds toa sequence-independent DNA:RNA duplex and does not bind to the nucleicacid molecule of interest or analyte of interest prior to the formationof the duplex. In one aspect, the antibody is S9.6.

In one aspect of embodiments provided herein directed to a system fordetecting an analyte of interest, the analyte is a polypeptide. In someaspects, the capture probe is an antibody that specifically binds to thepolypeptide.

In some aspects of any of the embodiments provided herein, the captureprobe is covalently coupled to the surface of the optical sensor oroptical ring resonator.

In another aspect of any of the embodiments provided herein, the opticalsensor or optical ring resonator comprises a waveguide structure. Invarious aspects, the optical sensor or optical ring resonator has anoutput portion configured to output an optical signal. In some aspects,said optical sensor or optical ring resonator has a first optical statewhen said capture probe binds to the analyte of interest forming saidcomplex and said antibody binds said complex, and wherein the opticalsensor or optical ring resonator has a second state when said antibodydoes not bind to said complex, the optical output yielding differentoutputs when said optical sensor or optical ring resonator in said firstand second optical states. In another aspect, the optical sensor oroptical ring resonator comprises an input and an output portion eachcomprising portions of a waveguide. In several aspects, the opticalsensor or optical ring resonator comprises an input waveguide and anoutput waveguide having optical coupling region therebetween configuredto increase coupling of a wavelength component from said input waveguideto said output waveguide when said capture probe binds to the analyte ofinterest forming said complex and said antibody binds to said complex.

In certain aspects of any of the aforementioned embodiments, saidoptical sensor or optical ring resonator is integrated on an integratedoptical chip comprising optical waveguides.

In various aspects of embodiments provided herein including an opticalsensor, the optical sensor comprises a resonator. In several aspects,the resonator has a resonant wavelength that shifts when said captureprobe binds to the analyte of interest forming said complex and saidantibody binds to said complex. In various aspects, the optical sensorcomprises a waveguide structure. Additionally in several aspects, theoptical sensor comprises a resonator formed from a closed loop or ringresonator, such as a racetrack resonator. In some aspects, saidclosed-loop resonator comprises a waveguide structure.

In certain aspects of any of the embodiments provided herein, theantibody increases the sensitivity of the optical sensor or optical ringresonator in detecting the nucleic acid molecule of interest or analyteof interest when the antibody binds to the duplex or complex.

In several aspects of any of the embodiments provided herein, theantibody amplifies the optical property that is altered when theantibody binds to the duplex or complex.

Several embodiments relate to a method for detecting a nucleic acidmolecule of interest in a sample including providing an optical sensorcomprising a nucleic acid capture probe attached to a surface of theoptical sensor, wherein the capture probe is capable of hybridizing tothe nucleic acid molecule of interest to form a duplex; applying asample for which the presence or absence of the nucleic acid molecule ofinterest is to be determined to the optical sensor under conditions inwhich the nucleic acid molecule of interest, when present, and thecapture probe sequence-specifically hybridize to form a duplex;providing an antibody that specifically binds a duplex of nucleic acidmolecules, wherein binding between the antibody and the duplex of thecapture probe and nucleic acid molecule of interest alters an opticalproperty of the optical sensor; and determining the presence or absenceof the nucleic acid molecule of interest by detecting the alteredoptical property of the optical sensor.

In one aspect, the nucleic acid molecule of interest comprisesribonucleic acid (RNA). In another aspect, the optical sensor comprisesa ring resonator. In various aspects, said ring resonator comprises awaveguide structure.

Various embodiments are drawn to a method for detecting an analyte ofinterest in a sample including providing an optical ring resonatorcomprising a capture probe attached to a surface of the optical ringresonator, wherein the capture probe is capable of binding to theanalyte of interest to form a complex; applying a sample for which thepresence or absence of the analyte of interest is to be determined tothe optical ring resonator under conditions in which the analyte ofinterest, when present, and the capture probe bind to form a complex;providing an antibody that specifically binds to the complex or analyte,wherein binding between the antibody and the complex or the analyte,when the analyte is bound to the capture probe, alters an opticalproperty of the optical ring resonator; and determining the presence orabsence of the analyte of interest by detecting the altered opticalproperty of the optical ring resonator.

In one aspect, the analyte of interest comprises ribonucleic acid (RNA).In another aspect, the analyte of interest is a polypeptide. In someaspects, the capture probe is an antibody that specifically binds to thepolypeptide. In an additional aspect, the optical ring resonatorcomprises a waveguide structure.

Certain embodiments are drawn to a system for detecting a polypeptide ofinterest in a sample including an optical sensor; a first antibody thatspecifically binds to the polypeptide of interest, wherein the firstantibody is attached to a surface of the optical sensor; a secondantibody that specifically binds to the polypeptide of interest; and aparticle attached to the second antibody or a particle capable ofbinding the second antibody, wherein said optical sensor has an opticalproperty that is altered when said second antibody is bound to saidpolypeptide of interest, when said polypeptide of interest is bound tothe first antibody, such that said optical sensor is configured to sensesaid second antibody combined with said polypeptide bound to said firstantibody, and the particle is adapted to amplify the optical propertythat is altered.

Several embodiments relate to a system for detecting a polypeptide ofinterest in a sample including an optical sensor configured to resonateat a resonant wavelength; a light source capable of providing light atsaid resonant wavelength for the optical sensor; a first antibody thatspecifically binds to the polypeptide of interest, wherein the firstantibody is attached to a surface of the optical sensor; a secondantibody that specifically binds to the polypeptide of interest; and aparticle attached to the second antibody or a particle capable ofbinding the second antibody; and a detector, wherein said optical sensorhas an optical property that is altered when said second antibody bindsto said polypeptide bound to said first antibody such that said opticalsensor is configured to sense said second antibody combined with saidpolypeptide bound to the first antibody; the particle is adapted toamplify the optical property that is altered; and the detector iscapable of detecting the optical property that is altered. The lightsource may comprise in various embodiments a laser such as a tuneablelaser or broad band light source such as a superluminescent laser diode(SLED).

Various embodiments are drawn to a system for detecting an analyte ofinterest in a sample including an optical sensor; a capture probeattached to a surface of the optical sensor, wherein the capture probeis capable of binding to the analyte; an antibody capable ofspecifically binding to the analyte or a complex formed between theanalyte and the capture probe; and a particle attached to the antibodyor capable of binding to the antibody, wherein said optical sensor hasan optical property that is altered by said antibody bound to saidcomplex or analyte, when the analyte is bound to the capture probe, suchthat said optical sensor is configured to sense said antibody combinedwith said analyte or complex, and the particle is adapted to amplify theoptical property that is altered.

In certain aspects of any one of the preceding systems including aparticle, the particle comprises a bead, polypeptide, nanoparticle,semiconductor crystal, titanium-oxide crystal, or quantum dot. Inadditional aspects, the particle comprises an average diameter of atleast 1 nm. In further aspects, the bead comprises silicon, polystyrene,agarose, sepharose, metal, or metal-oxide. In another aspect, theparticle comprises a polypeptide of at least 200 Daltons (Da). Invarious aspects, the polypeptide comprises myc, FLAG, GST, MBP, GFP, orbeta-gal. In a further aspect, the polypeptide comprises Protein A,Protein G, or a combination of Protein A and Protein G, and is capableof binding to the antibody. In yet another aspect, the polypeptidecomprises streptavidin and the antibody comprises biotin.

In certain aspects of the embodiments provided herein drawn to a systemfor detecting an analyte of interest in a sample including a particleattached to the antibody or capable of binding to the antibody, theanalyte is a polypeptide. In several aspects, the capture probe is anantibody that specifically binds to the polypeptide. In another aspect,the capture probe comprises an aptamer that specifically binds to thepolypeptide. In a further aspect, the capture probe comprises a proteinthat binds to the polypeptide.

In various aspects of the embodiments provided herein drawn to a systemfor detecting an analyte of interest in a sample including a particleattached to the antibody or capable of binding to the antibody, theanalyte is a nucleic acid. In some aspects, the nucleic acid comprisesribonucleic acid (RNA). In various aspects, the capture probe comprisesa DNA oligonucleotide. In several aspects, the DNA oligonucleotide iscomplementary to the nucleic acid. In various aspects, the DNAoligonucleotide comprises a modified DNA nucleotide, such as a lockednucleic acid (LNA) or universal base.

In other aspects, the capture probe comprises an RNA oligonucleotide. Insome aspects, the RNA oligonucleotide is complementary to the nucleicacid. In various aspects, the RNA oligonucleotide comprises a modifiedRNA nucleotide, such as a locked nucleic acid (LNA) or universal base.

In various aspects of the embodiments herein drawn to systems includinga particle attached to an antibody or capable of binding to an antibody,the capture probe is covalently coupled to the surface of the opticalsensor.

In various aspects of the embodiments herein drawn to systems includinga particle attached to an antibody or capable of binding to an antibody,the optical sensor comprises a waveguide structure. In other aspects,the optical sensor has an output portion configured to output an opticalsignal. In some aspects, said optical sensor has a first optical statewhen said capture probe binds to the analyte of interest forming saidcomplex and said antibody binds said complex, and wherein the opticalsensor has a second state when said antibody does not bind to saidcomplex, the optical output yielding different outputs when said opticalsensor in said first and second optical states.

In a further aspect, the optical sensor comprises an input and an outputportion each comprising portions of a waveguide. In several aspects, theoptical sensor comprises an input waveguide and an output waveguidehaving optical coupling region therebetween configured to increasecoupling of a wavelength component from said input waveguide to saidoutput waveguide when said capture probe binds to the analyte ofinterest forming said complex and said antibody binds to said complex.

In various aspects of the embodiments herein drawn to systems includinga particle attached to an antibody or capable of binding to an antibody,said optical sensor is integrated on an integrated optical chipcomprising optical waveguides. In further aspects, the optical sensorcomprises a resonator. In some aspects, said resonator has a resonantwavelength that shifts when said capture probe binds to the analyte ofinterest forming said complex and said antibody binds to said complex.In another aspect, the optical sensor comprises a waveguide structure.In various aspects, the optical sensor comprises a ring resonator. Insome aspects, said ring resonator comprises a waveguide structure.

Various embodiments are directed to a method for detecting a polypeptideof interest in a sample including providing an optical sensor comprisinga first antibody attached to a surface of the optical sensor, whereinthe first antibody specifically binds to the polypeptide of interest;applying a sample for which the presence or absence of the polypeptideof interest is to be determined to the optical sensor, under conditionsin which the polypeptide of interest, when present, and the firstantibody bind; providing a second antibody that specifically binds thepolypeptide of interest, wherein binding between the second antibody andthe polypeptide of interest, when bound to the first antibody, alters anoptical property of the optical sensor; providing a particle attached tothe second antibody or a particle capable of binding the secondantibody, wherein the particle amplifies the optical property that isaltered; and determining the presence or absence of the polypeptide ofinterest by detecting the altered optical property of the opticalsensor.

In one aspect, the optical sensor comprises a ring resonator. In severalaspects, said ring resonator comprises a waveguide structure.

Certain embodiments relate to a method for detecting an analyte ofinterest in a sample including providing an optical sensor comprising acapture probe attached to a surface of the optical sensor, wherein thecapture probe is capable of binding to the analyte of interest to form acomplex; applying a sample for which the presence or absence of theanalyte of interest is to be determined to the optical sensor, underconditions in which the analyte of interest, when present, and thecapture probe bind to form a complex; providing an antibody thatspecifically binds to the complex or analyte, wherein binding betweenthe antibody and the complex or the analyte, when the analyte is boundto the capture probe, alters an optical property of the optical sensor;providing a particle attached to the antibody or a particle capable ofbinding the antibody, wherein the particle amplifies the opticalproperty that is altered; and determining the presence or absence of theanalyte of interest by detecting the altered optical property of theoptical sensor.

In one aspect, the analyte of interest comprises ribonucleic acid (RNA).In another aspect, the optical sensor comprises a ring resonator. Insome aspects, said ring resonator comprises a waveguide structure.

In various embodiments described herein, the particle comprises metal.In particular, the particle may comprise gold. The particle maycomprises silver. In some embodiments the particle comprise dielectric.The particle may comprise a polymer. The particle may comprise a corethat is coated. In some embodiments, the coating provides opticalproperties (e.g., high refractive index) and/or may assist inapplication of a capture probe. The particle may be magnetic. In someembodiments, the particle comprises a magnetic core and may have anoverlayer or coating. Magnetic properties may be useful in processingthe particles. Accordingly, the particle can comprise a gold bead, asilver bead, a dielectric bead, a polymer bead, a magnetic bead or abead with a magnetic core. The bead may include a core and coating.

Several embodiments are drawn to detecting and/or measuring theconcentration of an analyte of interest in a sample using the systemsdescribed above, which can provide for real-time multiplex detection andmeasurement of low abundance biomolecules with high sensitivity andspecificity. It is possible to detect and/or measure binding-inducedshifts in the resonance wavelength resulting from individual bindingevents in real-time with the systems of several embodiments. In someembodiments, such binding events detectable in real-time include a“primary” binding event between an analyte of interest (with or withouta pre-bound particle) and a capture probe, a “secondary” binding eventbetween an antibody (with or without a pre-bound particle) and theanalyte of interest already bound to the capture probe, a “secondary”binding event between an antibody (with or without a pre-bound particle)and a duplex or complex formed between the analyte and capture probe, a“secondary” binding event between a particle and the analyte of interestalready bound to the capture probe (e.g. wherein the capture probecomprises an antigen and the analyte of interest is an antibody againstthe antigen), or a “tertiary” binding event between a particle andantibody already bound to the optical sensor via a “secondary” bindingevent. In some aspects, a plurality of the same type of particle, suchas a universal particle, can be used in a “tertiary” binding event. Incertain aspects, the plurality of the same type of particle can be usedin a multiplex format.

Various embodiments are directed to a system for detecting an analyte ofinterest in a sample, wherein the system includes a substrate, anoptical sensor disposed on said substrate, said optical sensor includingat least one waveguide, a first ring resonator, and a second ringresonator, wherein said at least one waveguide and said first and secondring resonators in optical communication with each other such that lightpropagating in the at least one waveguide can propagate to said firstand second ring resonators. The optical sensor, for example, one or moreof the at least one waveguide, the first ring resonator, and the secondring resonator may have a capture probe, for example, to capture ananalyte of interest.

In some embodiments, the first ring resonator and the second ringresonator are cascaded. In some embodiments, the first ring resonatorand the second ring resonator have substantially the same optical pathlength and resonant wavelengths.

In some embodiments, neither the first ring resonator is between the atleast one waveguide and the second ring resonator nor the second ringresonator is between the at least one waveguide and the first ringresonator. In some embodiments, the first ring resonator and the secondring resonator have different sizes and resonant wavelengths. In someembodiments, the first ring resonator and the second ring resonator havedifferent capture probes for capturing different analytes.

In some embodiments, the system further includes a waveguide structurethat is not a ring resonator disposed between the first and second ringresonators. In some embodiments, the first ring resonator and the secondring resonator form part of a Vernier resonator configuration. In someembodiments, the first ring resonator and second ring resonator havedifferent sizes and resonant wavelengths.

In various embodiments as described herein, beads or other particles maybe used to provide an amplifying effect on the signal. Other techniquessuch as those described herein may also be used to provide amplifyingeffects.

Various embodiments are directed to a system for detecting an analyte ofinterest in a sample, wherein the system includes a light source; awaveguide structure having a capture probe configured to bind with theanalyte of interest, said waveguide structure having an input forreceiving light from the light source such that light from the lightsource is guided in the waveguide structure; at least one particle thatis disposed in sufficient proximity to the waveguide structure when saidanalyte of interest binds with the capture probe such that at least aportion of said light guided within said light guide is scattered out ofthe light guide; and a detector for detecting at least a portion of saidlight propagating within the waveguide structure that is scattered outof the waveguide structure by the at least one particle.

In some embodiments, the waveguide structure comprises a ring resonator.

In some embodiments, the light source comprises a tunable laser.

In some embodiments, the light source comprises a super-luminescentdiode.

In some embodiments, the detector comprises a detector array.

In some embodiments, the system further includes scanning optics forreceiving light from said light source and directing said light to saidwaveguide structure. In some embodiments, said scanning optics aredisposed in a light path between said waveguide structure and saiddetector such that said scanning optics receive light scattered by saidparticles and directs said light to said detector.

Without being bound by theory, resonance wavelengths on the opticalsensor of several embodiments are sensitive to the local refractiveindex. Biomolecular binding events that increase the refractive index atthe sensor surface of various embodiments can be observed as an increasein the resonant wavelengths of the optical sensor. Similar to a sandwichassay format in which an antigen is first bound by asubstrate-immobilized primary capture agent and then recognized by asecondary capture agent, the systems of several embodiments include acapture probe (analogous to a sandwich assay primary capture agent) andan antibody (analogous to a sandwich assay secondary capture agent).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of a system for detecting an analytecomprising a light source that may include a light source (e.g. atunable light source or a broad band light source), an optical sensor,and an optical detector.

FIG. 2 shows a schematic diagram of an optical sensor comprising awaveguide and a ring resonator. FIG. 2 schematically illustrates therange of wavelengths that may be input into the optical sensor and theresultant spectral output of the optical sensor. A decrease in theoptical output at the resonance frequency of the ring resonator isvisible in the output spectrum shown.

FIG. 3 shows a cut-away view of the optical sensor comprising awaveguide and a ring resonator.

FIG. 4 is a perspective view an optical sensor such as shown in FIG. 3.

FIG. 5 is a cross-section through the waveguide and ring resonator shownin FIG. 4 along the line 5-5.

FIG. 6 is a cut-away view of a waveguide schematically showing anintensity distribution having an evanescent tail extending outside thewaveguide where an element such as a molecule or particle may be locatedso as to affect the index of refraction of the waveguide.

FIG. 7A is a cross-section through a waveguide such as a silicon stripwaveguide having a silicon dioxide layer thereon.

FIG. 7B is a cross-section through a waveguide such as a silicon ribwaveguide having a silicon dioxide layer thereon.

FIGS. 8A and 8B are schematic top views of optical sensors comprisingoval-shaped ring resonators.

FIG. 8C is a schematic top view of an optical sensor comprising atriangular-shaped ring resonator.

FIG. 8D is a schematic top view of an optical sensor comprising a ringresonator having an irregular shape.

FIG. 8E is a schematic top view of an optical sensor comprising a pairof waveguides having a ring resonator therebetween. This configurationmay be referred to as a drop configuration.

FIG. 8F is a schematic top view of an optical sensor comprising awaveguide and two cascaded ring resonators.

FIG. 8G is a schematic top view of an optical sensor comprising awaveguide and two ring resonators of different size disposedsubstantially parallel to the waveguide.

FIG. 8H is a schematic top view of an optical sensor comprising two ringresonators of different size alternated between three substantiallyparallel linear waveguides.

FIG. 9 schematically illustrates a plurality of optical sensors on achip and an apparatus that provides light to the chip and detects lightoutput from the chip.

FIG. 10 is a perspective view of light coupled into a waveguide on achip using a grating coupler and light coupled out of a waveguide on achip using a grating coupler, for example, to provide input to andcollect output from an optical sensor on the chip.

FIG. 11 is a top view schematically illustrating a chip having input andoutput couplers connected to waveguide optical sensors comprising ringresonators. The chip further includes flow channels for flowing solutionacross the waveguide optical sensors and in particular the ringresonators. Input ports provide access to the flow channels. The chipfurther comprises identification markers to facilitate identification ofthe different optical sensors.

FIG. 12A shows a schematic diagram of the S9.6 amplification assay. Amicroring is covalently modified with ssDNA capture probes on itssurface. The sensor is exposed to a solution containing the targetmiRNA, after which the S9.6 antibody is flowed across the surface,binding only to DNA:RNA heteroduplexes. FIG. 12B is a graph showing thesignal response from 3 separate microrings corresponding to theschematic in FIG. 12A.

FIG. 13 is a graph showing the amplification response to microringssaturated with a 10mer RNA, 20mer RNA, or 40mer RNA bound to a 40merssDNA capture probe in terms of relative wavelength shift over time.

FIG. 14 shows S9.6 amplification response towards: 100 nM miR-24-1 witha 22mer capture probe, 100 nM miR-24-1 with a 54mer capture probe, 1 nMmiR-24-1 with a 22mer capture probe, and 1 nM miR-24-1 with a 54mercapture probe in terms of relative wavelength shift over time.

FIG. 15A is a graph showing the real time response of S9.6 amplificationtowards a DNA:DNA homoduplex and a DNA:RNA heteroduplex in terms ofrelative wavelength shift over time. FIG. 15B is a graph showing theresponse of S9.6 amplification towards a DNA:DNA homoduplex and aDNA:RNA heteroduplex under saturation conditions in terms of relativewavelength shift over time.

FIGS. 16-1 to 16-4 are graphs showing simultaneous amplification ofmiRNA targets in terms of relative wavelength shift over time. Onlythose channels containing complementary capture probes and target miRNAselicit an S9.6 response, allowing multiplexed miRNA analysis.

FIG. 17 shows an overlay of the signal responses achieved for eachconcentration of target miRNA: miR-21 (FIG. 17A), miR-24-1 (FIG. 17B),miR-16 (FIG. 17C), and miR-26a (FIG. 17D). Concentrations utilized were40 nM, 10 nM, 2.56 nM, 640 pM, 160 pM, 40 pM, 10 pM, and a blank (withthe exception of miR-16, which did not contain the 40 pM and 10 pMcalibration points).

FIG. 18 shows calibration curves for the S9.6 response for miR-16 (FIG.18A), miR-21 (FIG. 18B), miR-24-1 (FIG. 18C), and miR-26a (FIG. 18D)that represent the logistic fits to the data points. Error barsrepresent ±1 standard deviation for between 4 and 12 independentmeasurements at each concentration.

FIG. 19 is a bar graph showing a comparison of the concentrations foreach of the four target miRNAs (miR-16, miR-21, miR-24-1, and miR-26a)in total mouse brain RNA.

FIG. 20 is a graph showing the S9.6 response in terms of relativewavelength shift over time to varied ssDNA capture probe concentrationswith a constant miR-24-1 target concentration (40 nM).

FIG. 21 is a graph showing tertiary binding of streptavidin quantum dotsto a biotinylated secondary antibody in terms of relative wavelengthshift over time. The tertiary binding lowers the detection limit for theanalyte IL-2 by at least 10-fold down to the low 100s of fM.

FIG. 22A is a graph showing real-time response of protein G-conjugatedpolystyrene beads binding to an array of antibody-functionalizedmicroring resonators. The discrete jumps in relative resonancewavelength shift can be attributed to individual binding events ofeither single beads or bead aggregates. FIG. 22B is a scanning electronmicroscopy (SEM) image stitched from four high resolution images, whichallows enumeration of beads bound to a given microring. Only beadsdirectly contacting the ring within the evanescent field are counted.FIG. 22C is a plot of resonance wavelength shifts versus number of boundbeads, which illustrates a linear trend providing evidence thatindividual, biomolecularly directed bead binding events can be observedusing a microring resonator.

FIG. 23 is a graph showing real-time response of an array of twelvebiotin-functionalized microring resonators to avidin-coated latex beads.The resonances show discrete jumps in resonance frequence that can beattributed to individual bead binding events.

FIGS. 24A-F are graphs showing signal enhancement using secondaryantibody and tertiary bead-based detection applied to the detection ofthe serum cancer biomarker alpha-fetoprotein in terms of relativewavelength shift over time (FIGS. 24A-C) or over concentration (FIGS.24D-F). Label-free primary binding event detection is shown in FIGS. 24Aand 24D, antibody binding to bound antigen secondary binding eventdetection is shown in FIGS. 24B and 24E, and beads binding to boundantibodies tertiary binding event detection is shown in FIGS. 24C and24F.

FIG. 25 is a schematic diagram of an auto-antibody multiplex opticalring detection system.

FIGS. 26A-D are graphs showing correlation between the optical ringdetection system and ELISA for the detection of the auto-antibodies toJo-1 (FIG. 26A), SSA and SSB (FIG. 26B), Smith (FIG. 26C), and Scl-70(FIG. 26D).

FIG. 27 is a bar graph comparing the sensitivity of the optical ringdetection system compared to ELISA in detecting the auto-antibodies toJo-1, SSA and SSB, Smith, and Scl-70.

FIG. 28 is a panel of graphs plotting wavelength shift over time on 5chips, each having a microring to detect one of the Jo-1, SSA and SSB,Smith, and Scl-70 auto-antibodies in control sera sample known to bepositive for 1 or 2 of the autoantibodies. The results show no-crosstalk between the microrings.

FIG. 29A schematically illustrates an apparatus for interrogatingoptical scattering from optical resonators on a chip by using scanningmirrors. FIG. 29B schematically illustrates an apparatus forinterrogating scattering from optical resonators on a chip by usingimaging optics that form an image of a portion of the chip containing aplurality of such resonators onto a detector array.

FIG. 30A is a schematic showing a process of binding a particle attachedto horse radish peroxidase (HRP) to an analyte of interest, which isalready bound to an antibody capture probe, and precipitating thesubstrate 3,3′-diaminodibenzidine (DAB) onto the microring. FIG. 30B isa schematic showing a process of attaching an antibody capture probe toa microring, binding an analyte to the antibody capture probe, andbinding a particle to the analyte. FIG. 30C is a schematic showing aprocess of pre-mixing an analyte with a particle to form a complex andbinding the complex to an antibody capture probe attached to amicroring. FIG. 30D is a schematic illustrating binding between anantibody attached to a particle and an analyte bound to an antibodycapture probe on a microring.

DETAILED DESCRIPTION

In contrast to existing analyte detection technologies, various systemsof several embodiments provided herein feature one or more of thefollowing: high detection sensitivity and specificity, scalability andmultiplex capacity, ability to analyze large analytes, and ability todetect or measure multiple individual analyte binding events inreal-time. Furthermore, the systems and methods of various embodimentsinvolve low sample volume in the microliter range and only a relativelysmall amount of hands-on time, and provide rapid time to results, whichare reproducible. Eliminating the drawbacks of current technologies, thesystems and methods of various embodiments are a major technologicalbreakthrough in analyte detection, surpassing the existing detectionplatforms for widespread applicability in diverse analytical settings.

Optical sensors, such as silicon photonic microring resonators, havehigh spectral sensitivity towards surface binding events between ananalyte of interest and an optical sensor modified with a probe forcapturing the analyte of interest (i.e. a capture probe). The systems ofseveral embodiments are based on refractive index-based sensing schemesin which the mass of bound analytes, potentially in combination withother factors such as capture probe affinity and surface density,contributes to the observed signal and measurement sensitivity.

Analytes, such as proteins, that are simultaneously low in abundance andhave a lower molecular weight are often very difficult to detect.Several embodiments relate to employing a more massive antibody toamplify the signal arising from the initial primary binding eventbetween the analyte and capture probe. Based on the present discoverythat a remarkable femptomolar (10⁻¹⁵) range of detection sensitivity canbe achieved, various embodiments relate to employing a particle tofurther amplify the signal arising from the primary binding event and/orthe signal arising from the secondary binding event of the “secondary”antibody. In certain embodiments, it is possible to improve both thesensitivity and/or the specificity of analyte detection assays, allowingfor quantitative sensing in complex sample matrices.

One important class of analytes, microRNAs (miRNAs), are expressed atlow levels in many organisms but nevertheless have important cellularroles and are associated with several diseases. miRNAs have becomeimportant biomarkers for a variety of diseases and conditions, butexisting technologies lack the sensitivity to adequately detect ormeasure them due to their low abundance.

The systems of several embodiments herein can improve the sensitivity ofdetecting miRNA analytes in a rapid, multiplexed, and high-throughputdetection format in real-time. The systems of several embodiments hereincan detect microRNA at concentrations as low as 10 pM (350 attomoles)with a rapid time-to-result. The simplicity and widespread applicabilityof various of these embodiments make them an useful tool forhigh-throughput, multiplexed miRNA analysis, as well as a range of otherRNA based detection applications.

It will be understood that as used herein, the singular forms “a,” “an”,and “the” include plural referents unless indicated to the contrary.Also, it will be understood that the term “detecting” an analyte as usedherein also includes measuring the amount or concentration of an analytebecause the systems and methods of various embodiments can provide bothqualitative and quantitative detection, which can include measurement ofa small number or even individual binding events in real-time.

Optical Sensing

Analyte detection can be accomplished using an optically based system100 such as shown schematically in FIG. 1. The system 100 includes alight source 102, an optical sensor 104, and an optical detector 106. Invarious embodiments, the light source 102 outputs a range ofwavelengths. For example, the light source 102 may be a relativelynarrow-band light source that outputs light having a narrow bandwidthwherein the wavelength of the light source is swept over a region manytimes the bandwidth of the light source. This light source 102 may, forexample, be a laser. This laser may be a tunable laser such that thewavelength of the laser output is varied. In some embodiments the laseris a diode laser having an external cavity. This laser need not belimited to any particular kind and may, for example, be a fiber laser, asolid state laser, a semiconductor laser or other type of laser or lasersystem. The laser itself may have a wavelength that is adjustable andthat can be scanned or swept. Alternatively, additional opticalcomponents can be used to provide different wavelengths. In someembodiments, the light source outputs light having a wavelength forwhich the waveguide structure is sufficiently optically transmissive. Insome embodiments, the waveguide structure is within a sample medium suchas an aqueous medium and the light source outputs light having awavelength for which the medium is substantially optically transmissivesuch that resonance can be reached in the optical resonator.Additionally, in some embodiments, the light source output has awavelength in a range where the analyte (e.g., molecules) of interest donot have a non-linear refractive index. Likewise, in variousembodiments, the light source 102 may be a coherent light source andoutput light having a relatively long coherence length. However, invarious embodiments, the light source 102 may be a coherent light sourcethat outputs light having a short coherence length. For example, incertain embodiments, a broadband light source such as asuper-luminescent light emitting diode (SLED) may be used. In suchcases, the wavelength need not be swept.

The light source 102 provides light to the optical sensor 104. The lightsource 102 may be controlled by control electronics. These electronicsmay, for example, control the wavelength of the light source, and inparticular, cause the light source 102 to sweep the wavelength of theoptical output thereof. In some embodiments, a portion of the lightemitted from the light source 102 is sampled to determine, for example,the emission wavelength of the light source.

In some embodiments, the optical sensor 104 comprises a transducer thatalters the optical input based on the presence and/or concentration ofthe analyte to be detected. The optical sensor 104 may be a waveguidestructure. The optical sensor 104 may be an integrated optical deviceand may be included on a chip. The optical sensor 104 may comprisesemiconductor material such as silicon. The optical sensor 104 may be aninterferometric structure (e.g., an interferometer) and produce anoutput signal as a result of optical interference. The optical sensor104 may be included in an array of optical sensors 104.

The optical detector 106 detects the optical output of the sensor 104.In various embodiments, the optical detector 106 comprises a transducerthat converts an optical input into an electrical output. Thiselectrical output may be processed by processing electronics to analyzethe output of the sensor 104. The optical detector 106 may comprise aphotodiode detector. Other types of detectors 106 may be employed.Collection optics in an optical path between the sensor 104 and thedetector 106 may facilitate collection of the optical output of thesensor 104 and direct this output to the detector 106. Additional opticssuch as mirrors, beam-splitters, or other components may also beincluded in the optical path from the sensor 104 to the detector 106.

In various embodiments, the optical sensor 104 is disposed on a chipwhile the light source 102 and/or the optical detector 106 are separatefrom the chip. The light source 102 and optical detector 106 may, forexample, be part of an apparatus comprising free space optics thatinterrogates the optical sensors 106 on the chip, as will be discussedin more detail below.

In various embodiments, a solution 108 such as an analyte solution isflowed past the optical sensor 104. The detector 106 detects modulationin an optical signal from the optical sensor 104 when an analyte ofinterest is detected.

Ring resonators offer highly sensitive optical sensors that can beprepared so as to detect analytes. The operation of a ring resonator isshown in connection with FIG. 2. In this configuration, the opticalsensor 104 comprises an input/output waveguide 202 having an input 204and an output 206 and a ring resonator 208 disposed in proximity to aportion of the input/output waveguide 202 that is arranged between theinput 204 and the output 206. The close proximity facilitates opticalcoupling between the input/output waveguide 202 and the ring resonator208, which is also a waveguide. In this example, the input/outputwaveguide 202 is linear and the ring resonator 208 is circular such thatlight propagating in the input/output waveguide 202 from the input 204to the output 206 is coupled into the ring resonator 208 and circulatestherein. Other shapes for the input/output waveguide 202 and ringresonator 208 are also possible.

FIG. 2 shows an input spectrum 210 to represent that the light injectedinto the waveguide input 204 includes a range of wavelengths, forexample, from a narrow band light source having a narrow band peak thatis swept over time (or from a broadband light source such as asuper-luminescent diode). Similarly, an output spectrum 212 is shown atthe waveguide output 206. A portion of this output spectrum 212 isexpanded into a plot of intensity versus wavelength 214 and shows a dipor notch in the spectral distribution at the resonance wavelength, λ₀,of the ring resonator 208.

Without subscribing to any particular scientific theory, light“resonates” in the ring resonator when the number of wavelengths aroundthe ring (e.g. circumference) is exactly an integer. In this example,for instance, at particular wavelengths, light circulating in the ringresonator 208 is at an optical resonance when

mλ=2 πrn   Eq. 1

where m is an integer, λ is the wavelength of light, r is the ringradius, and n is the refractive index. In this resonance condition,light circulating in the ring interferes with light propagating withinthe linear waveguide 202 such that optical intensity at the waveguideoutput 206 is reduced. Accordingly, this resonance will be measured asan attenuation in the light intensity transmitted down the linearwaveguide 202 past the ring resonator 208 as the wavelength is swept bythe light source in a manner such as shown in the plot 214 of FIG. 2.

Notably, the plot 214 in FIG. 2 shows the dip or notch having a width,au as measured at full width half maximum (FWHM) and an associatedcavity Q or quality factor, Q=λ₀/δv. The ring resonator 208 produces arelatively high cavity Q and associated extinction ratio (ER) thatcauses the optical sensor 104 to have a heightened sensitivity.

A perspective view of the optical sensor 104 comprising a linearwaveguide 202 and a ring resonator 208 is shown in FIG. 3. Both arewaveguide structures as is this optical sensor 104. The linear waveguide202 and the ring resonator 208 are disposed on a substrate 302 with alower cladding layer 304 therebetween. Other configurations arepossible, for example, other layers may be added (or removed) orpatterned differently. This portion of the substrate 302 having thelinear waveguide 202 and ring resonator 208 formed thereon may be partof a larger integrated optical chip.

A drawing of an example biosensor waveguide structure comprising alinear waveguide 202 and a ring resonator 208 is also shown in FIG. 4.An upper cladding 402 is disposed over most of the area shown. However,a window 404 (here annular in shape) is included in the upper cladding402 and provides exposure to portions of the linear waveguide 202 andthe ring resonator 208. An analyte solution can thereby be flowed acrossthe linear waveguide 202 and ring resonator 208 and permitted tointeract therewith. The upper cladding 402 limits the exposure of theintegrated waveguide structure to the analyte solution.

A cross-section through the line 5-5 shown in FIG. 4 is presented inFIG. 5. The cross-section shows the linear waveguide 202 and the ringresonator 208 disposed over the lower cladding 304 and substrate 302.The upper cladding 402 is also illustrated. As discussed above, openingsor windows 404 in the upper cladding 402 provide access for the analytesolution to the linear waveguide 202 and ring resonator 208. A flowchannel 502 (shown schematically by an arrow) for the analyte solutionis also illustrated.

As is well known, light propagates within waveguides via total internalreflection. The waveguide supports modes that yield a spatially varyingintensity pattern across the waveguide. A cross-section of a waveguide602 shown in FIG. 6 illustrates an example intensity distribution 604. Aplot 606 of the intensity distribution at different heights is providedadjacent the waveguide structure 602. As illustrated, a portion 608 ofthe electric field and optical energy referred to as the evanescent“tail” lies outside the bounds of the waveguide 602. The length of thisfield 608, as measured from the 1/e point, is between 50 and 150 nm,e.g. about 100 nm in some cases. An object 610 located close to thewaveguide 602, for example, within this evanescent field length affectsthe waveguide. In particular, objects 610 within this close proximity tothe waveguide 602 affect the index of refraction of the waveguide. Theindex of refraction, n, can thus be different when such an object 610 isclosely adhered to the waveguide 602 or not. In various embodiments, forexample, the presence of an object 610 increases the refractive index ofthe waveguide 602. In this manner, the optical sensor 104 may beperturbed by the presence of an object 610 in the vicinity of thewaveguide structure 602 thereby enabling detection. In variousembodiments, the size of the particle is about the length (e.g. 1/edistance) of the evanescent field to enhance interaction therebetween.

In the case of the ring resonator 208, an increase in the refractiveindex, n, increases the optical path length traveled by lightcirculating about the ring. Longer wavelengths can resonate in theresonator 208 and, hence, the resonance frequency is shifted to a lowerfrequency. The shift in the resonant wavelengths of the resonator 208can therefore be monitored to determine if an object 610 has locateditself within close proximity to the optical sensor 104 (e.g., the ringresonator 208 and/or a region of the linear waveguide 202 closest to thering resonator). A binding event, wherein an object 610 binds to thesurface of the optical sensor 104 can thus be detected by obtaining thespectral output 212 from the waveguide output 206 and identifying dipsin intensity (or peaks in attenuation) therein and the shift of thesedips in intensity.

In various embodiments, the waveguide 602, e.g., the linear waveguide202 and/or the ring resonator 208 comprise silicon. In some embodiments,the surface of the waveguide 602 may be natively passivated with silicondioxide. As a result, standard siloxane chemistry may be an effectivemethod for introducing various reactive moities to the waveguide 602,which are then subsequently used to covalently immobilize biomoleculesvia a range of standard bioconjugate reactions.

Moreover, the linear waveguide 202, ring resonator 208, and/oradditional on-chip optics may be easily fabricated on relatively cheapsilicon-on-insulator (SOI) wafers using well established semiconductorfabrication methods, which are extremely scalable, cost effective, andhighly reproducible. Additionally, these devices may be easilyfabricated and complications due to vibration are reduced when comparedto “freestanding” cavities. In one example embodiment, 8″ SOI wafers mayeach contain about 40,000 individually addressable ring resonators 208.One advantage of using silicon-based technology is that variousembodiments may operate in the Si transparency window of around 1.55 μm,a common optical telecommunications wavelength, meaning that lasers anddetectors are readily available in the commercial marketplace asplug-and-play components.

FIGS. 7A and 7B show cross-sectional views of two example waveguides602, each having a thin layer 702 such as of silicon dioxide on the topof the waveguides 602. In various embodiments, the thickness of thinlayer 702 is substantially less than the length of the evanescent field608, so that, for example, some of the evanescent field reaches thebinding site, although thicker or thinner layers are possible. Asdiscussed above, in some cases, this thin layer 702 facilitatesdeposition of a binding probe layer on the surface of the waveguidesensor 104. This binding probe layer may bind with analytes to bedetected. Such a binding event would cause the index of refraction ofthe waveguide resonator 208 to increase and the resonance frequencythereof to shift in a manner that is detectable by the optical detector106.

The waveguides 602 in FIGS. 7A and 7B are often referred to as strip andrib waveguides. Other types of waveguides, such as for example,strip-loaded waveguides can be used. Lower cladding 304 lies beneath thewaveguides 602. As discussed above, in some embodiments, the waveguides602 are formed from a silicon-on-insulator chip, wherein the silicon ispatterned to form the waveguides 602 and the insulator beneath providesthe lower cladding 304. In many of these embodiments, thesilicon-on-insulator chip further includes a silicon substrate. Detailson the fabrication of silicon biosensor chips can be found in Washburn,A. L., L. C. Gunn, and R. C. Bailey, Analytical Chemistry, 2009, 81(22):p. 9499-9506, and in Bailey, R. C., Washburn, A. L., Qavi, A. J., Iqbal,M., Gleeson, M., Tybor, F., Gunn, L. C. Proceedings of SPIE—TheInternational Society for Optical Engineering, 2009, the disclosures ofwhich are hereby incorporated by reference in their entirety.

Although circularly-shaped ring resonators have been discussed above,the ring resonator 208 may have other shapes. FIGS. 8A through 8E showvarious examples of ring resonators 208. Oval or elliptically-shapedring resonators 802 are illustrated in FIGS. 8A and 8B. In FIG. 8A, theelliptically-shaped resonator 802A has a major axis parallel with thelinear waveguide 202. In FIG. 8B, the elliptically-shaped resonator 802Bhas a minor axis parallel with the linear waveguide 202. The oval orelliptically-shaped resonator 802 can be oriented differently as well.

A triangularly-shaped ring resonator 804 is shown in FIG. 8C. Thetriangularly-shaped ring resonator 804 has three linear segments 806.Three mirrors 808 are also included at the junction between the linearsegments 806. Additional segments 806 and mirrors 808 may be added tocreate different shapes.

FIG. 8D illustrates a ring resonator 810 having an arbitrary shape. Theshape of the resonator can be varied as desired.

In each of FIGS. 8A-8D, the ring resonators 802A, 802B, 804, 810 areshown in proximity to the linear waveguide 202 so as to provide opticalcoupling therebetween. In some cases for example, the distance, d,separating the linear waveguide 202 and the ring resonator 802A, 802B,804, 810, is about the size of the evanescent field in the linearwaveguide and the evanescent field in the ring resonator at the locationwhere the two waveguide structures are closest. Larger or smaller valuesmay be possible in other cases. Transfer of optical energy is providedvia overlap of the evanescent fields.

FIG. 8E shows a different configuration, which may be referred to as adrop configuration, wherein a ring resonator 812 is disposed betweenfirst and second waveguides 814 a and 814 b. Light (e.g. a wavelengthcomponent) may be directed into an input 816 of the first waveguide 814a and depending on the state of the ring resonator 812, may be directedto either an output 818 a of the first waveguide 814 a or an output 818b of the second waveguide 814 b. For example, for resonant wavelengths,the light may be output from the second waveguide 814 b instead of thefirst waveguide 814 a. The optical detector 106 may thus monitor shiftsin intensity peaks to determine the presence of an analyte of interestdetected by the optical sensor 104.

Various embodiments may incorporate more than one ring resonator. FIG.8F shows an example configuration wherein a first ring resonator 822 aand a second ring resonator 822 b are employed. In the embodiment shownin FIG. 8F, the first ring resonator 822 a and a second ring resonator822 b are cascaded or arranged in a series and in sufficiently closeproximity to interact with each other. The first and second ringresonators 822 a, 822 b are disposed with respect to a linearinput/output waveguide 202 such that the first ring resonator 822 a isbetween the input/output waveguide 202 and the second ring resonator 822b. The first ring resonator 822 a is a distance d from the linearinput/output waveguide 202 so as to be optically coupled together. Thesecond ring resonator 822 b is the same distance d from first ringresonator 822 a, also so as to be optically coupled together. Light maybe coupled from the input/output waveguide 202 into the first ringresonator 822 a as in FIGS. 8A-8D, and then into the second ringresonator 822 b. In various embodiments, the perimeter of the first ringresonator 822 a is equal to the perimeter of the second ring resonator822 b. In some embodiments, a cascade effect is produced when lighthaving a wavelength matching a resonance wavelength of both the firstand second ring resonators 822 a and 822 b is coupled from theinput/output waveguide 202 into the first ring resonator 822 a and theninto the second ring resonator 822 b. The optical transmission spectrum,graphed in output plot 214, will include a dip or notch at the resonantwavelength(s). In some embodiments, the cascaded resonators may decreasethe width of the dip or notch in the transmission spectrum and providethe output plot 214 with a more “box-like” or “flat” center and possiblysteeper falloff in comparison to having the first ring resonator 822 awithout the second ring resonator 822 b. Cascade effects in coupled ringresonators are discussed further in Little, B. E., Chu, S. T., Haus, H.A., Foresi, J., and Laine, J.-P., Microring Resonator Channel DroppingFilters, J. Lightwave Technology, 15, 998 (1997), the disclosure ofwhich is hereby incorporated by reference in its entirety.

Although two resonators are shown in FIG. 8F, more ring resonators maybe added. Additionally, the ring resonators may be positioneddifferently with respect to each other as well with respect to theinput/output waveguide 202. The resonators may also have different sizesand/or shapes. A drop configuration such as shown in FIG. 8E may also beused instead of having a single input/output waveguide 202. Combinationsof these different features are also possible.

FIG. 8G shows an example configuration of an embodiment wherein multiplering resonators are aligned along the length of and adjacent to theinput/output waveguide 202. The first ring resonator 822 c is disposed adistance d from the input/output waveguide 202. The second ringresonator 822 d is also disposed a distance d from the waveguide 202.Unlike FIG. 8F, the first ring resonator 822 c is not disposed betweenthe input/output waveguide 202 and second ring resonator 822 d.Similarly, the second ring resonator 822 d is not disposed between theinput/output waveguide 202 and first ring resonator 822 c. Both ringresonators 822 c and 822 d are disposed in proximity to the input/outputwaveguide 202, such that light can be coupled from the input/outputwaveguide to both the ring resonators 822 c and 822 d without needing topass through the other ring resonator first. Both ring resonators 822 cand 822 d are on the same side of the waveguide 202. The first ringresonator 822 c is disposed a distance greater than d from the secondring resonator 822 d. In various embodiments, this distance greater thand is longer than the evanescent field length 608 such that light is notcoupled directly from first ring resonator 822 c into second ringresonator 822 d, and vice versa. In various embodiments, the perimeterof the first ring resonator 822 c is unequal to the perimeter of thesecond ring resonator 822 d. Accordingly, the first ring resonator 822 chas a different resonant wavelength(s) than the second ring resonator822 d.

This example configuration may be used in conjunction with a broadspectrum light source, such as a super-luminescent light emitting diode(SLED) or an erbium amplifier running broadband, to simultaneouslydetect multiple analytes by interrogating the first ring resonator 822 cand the second ring resonator 822 d simultaneously. The broad spectrumlight source emits light that travels through waveguide 202. The firstring resonator 822 c may be associated with a first resonant wavelengthand a first analyte. The second ring resonator 822 d may be associatedwith second resonant wavelength and a second analyte. The presence ofthe first analyte may cause a shift in a notch in the transmissionspectrum output plot 214 at the first resonant wavelength when bound tothe first ring resonator 822 c, while the presence of the second analytemay cause a shift in a notch in the absorption spectrum output plot 214at the second different resonant wavelength when bound to the secondring resonator 822 d. Other configurations can be used. For example, atunable laser or other tunable light source may be used instead of abroadband light source and the wavelength of the output of the tunablelaser can be swept. Similarly, the first and second notches in thetransmission spectrums of the first and second ring resonators 822 c,822 d can be monitored to detect the presence of the first and secondanalytes respectively.

Although two resonators are shown in FIG. 8G, more ring resonators maybe added. Additionally, the ring resonators may be positioneddifferently with respect to each other as well as with respect to theinput/output waveguide 202. For example, the ring resonators may be onopposite sides of the input/output waveguide 202. As discussed above,the resonators may also have different sizes and/or shapes. A dropconfiguration such as shown in FIG. 8E may also be used instead ofhaving a single input/output waveguide. Combinations of these differentfeatures are also possible.

FIG. 8H depicts an example optical sensor 104 comprising a plurality ofring resonators and a plurality of waveguides that are not ringresonators arranged such that at least one of the ring resonators isbetween two of the non-ring resonator waveguide structures and at leastone of the non-ring resonator waveguide structures is disposed betweentwo of the ring resonators. A first ring resonator 822 e is disposedbetween a first input/output non-ring resonator waveguide 824 a and asecond “intermediate” non-ring resonator waveguide 824 b (both shown aslinear waveguides in FIG. 8H). The first ring resonator 822 e isdisposed a distance d from first waveguide 824 a and a distance d fromsecond intermediate waveguide 824 b. The optical sensor furthercomprises a second ring resonator 822 f disposed between the secondintermediate waveguide 824 b and a third “input/output” non-ringresonator waveguide 824 c (shown as a linear waveguide in FIG. 8H). Thesecond ring resonator 822 f is disposed a distance d from secondwaveguide 824 b and a distance d from third input/output waveguide 824c. In some embodiments, the first and second ring resonators 822 e, 822f are offset with respect to each other (e.g., along the length of thewaveguides 824 a, 824 b, 824 c).

In various embodiments, light may be directed into an input 826 of thefirst input/output waveguide 824 a, and, depending on the state of thefirst ring resonator 822 e and the wavelength of light, may be directedto either an output 828 a of the first waveguide 824 a, or may bedirected into second waveguide 824 b. For example, for the resonantwavelengths of the first ring resonator 822 e, the light may be coupledinto the second waveguide 824 b instead of being output from the firstwaveguide 824 a at output 828 a. Light coupled into the second waveguide824 b from the first ring resonator 822 e is directed to either anoutput 828 b of the second waveguide 824 b or into the third waveguide824 c, depending on the state of the third ring resonator 822 f. Forexample, for the resonant wavelengths of the third ring resonator 822 f,the light may be coupled into the third waveguide 824 c and then outputat output 828 c. In the case where the light source that directs lightinto the first input/output waveguide 826 comprises a broadband lightsource such as a super-luminescent diode that outputs a broadbandspectrum, the light referred to above may be a wavelength component ofthe broader spectrum.

In various embodiments, the perimeter of the first ring resonator 822 eis unequal to the perimeter of the second ring resonator 822 f, suchthat the Free Spectral Range (FSR) of the first ring resonator 822 e isslightly different from the FSR of the second ring resonator 822 f. Invarious embodiments, this configuration can produce Vernier effects.Light directed into the input 826 can pass through both the first ringresonator 822 e and the second ring resonator 822 f if it is of aresonant wavelength common to both the first ring resonator 822 e andthe second ring resonator 822 f. Two resonators with slightly differentFSRs have a large combined FSR, as their common resonant wavelengths arehighly separated in the wavelength spectrum. Accordingly, the passbandstransmitted from input 826 to output 828 c by this configuration arerelatively far apart in the wavelength spectrum as these passbandscoincide with the common resonant wavelengths of first ring resonator822 e and second ring resonator 822 f. Additionally, embodiments of thisconfiguration may have relatively narrow passband bandwidths. OpticalVernier effects are also discussed in Schwelb, O., The Vernier Principlein Photonics, 2011, the disclosure of which is hereby incorporated byreference in its entirety.

Other configurations can be used. A tunable laser or other tunable lightsource may be used as the input source and the wavelength of the outputof the tunable laser can be swept. Alternatively, a broadband lightsource such as a superluminescent diode may be used.

More ring resonators may be added. Additionally, the ring resonators maybe positioned differently with respect to each other as well as withrespect to the input/output waveguide 202. Likewise, more non-ringresonator waveguides may be added. As discussed above, the resonatorsmay also have different sizes and/or shapes. In some embodiments, thethird output 828 c or last non-ring resonator waveguide 824 c may beexcluded. Combinations of these different features are also possible.

Still other designs than those shown in FIGS. 8F-8H may be employed.Multiple resonators and/or waveguides may be placed in any desiredgeometric arrangement. Additionally, spacing between resonators and/orwaveguides may be varied as desired. Different features from FIG. 8A-8Hcan be combined in different ways. Still other configurations arepossible

Other geometries may possibly be used for the resonator, such as, forexample, microsphere, microdisk, and microtoroid structures. See, e.g.,Vahala, Nature 2003, 424, 839-846; and in Vollmer & Arnold, NatureMethods 2008, 5, 591-596, the disclosures of which are herebyincorporated by reference in their entirety.

Also, although linear waveguides 202 are shown in FIGS. 8A-8G asproviding access to the ring resonators 208 such as those shown by 802a, 802 b, 804, 810, 812, 822 a, 822 c, and 822 d, these waveguides neednot be restricted to plain linear geometry. In some examples, forinstance, these waveguides 202 may be curved or otherwise shapeddifferently.

Various embodiments of ring resonators and possibly other geometriesrepeatedly circulate light around, for example, their perimeter,dramatically increasing the optical path length. Furthermore,interference between photons circulating in the structure and thosetraversing the adjacent waveguide create a resonant cavity ofextraordinarily narrow spectral linewidth resulting in a high-Q device.The resulting resonance wavelengths are quite sensitive to changes inthe local refractive index. As discussed herein, this sensitivityenables the sensors to detect small masses.

In various embodiments as described herein, beads and other particlesmay be used to provide an amplifying effect on the signal. Othertechniques such as those described herein may also be used to provideamplifying effects.

One embodiment of an apparatus 900 for interrogating the optical sensors104 on a chip 902 is schematically illustrated in FIG. 9. The apparatus900 includes a laser light source 904, which may comprise a tunablelaser. The apparatus 900 further comprises a splitter 906 that directslight from the laser 904 along a first path 908 to a photodetector 910for calibration and along a second path 912 toward the chip 902. Astatic Fabry-Perot cavity or other wavelength resolving device 914 maybe included in the first path 908 to the photodetector 910 such that thephotodetector 910 can measure the relative power for differentwavelengths of the light output by the laser 904 and presumably providedto the optical sensors 104. The wavelength resolving device 914 mayestablish a reference wavelength that is known to be output from thelight source at a specific time. By additionally knowing the rate atwhich the wavelengths are swept, the wavelength output by the lightsource at different times is can be determined. Beam shaping optics,such as a collimator 916, may be included in the second optical path 912to adjust the shape of the beam as desired. This beam is directed toscanning mirrors 918 such that the beam may be scanned across the chip902. Focusing optics 920 are included to focus the beam onto the chip902.

The chip 902 includes input couplers 922 configured to couple the beampropagating in free space into the waveguides 202 on the chip. Theseinput couplers 922 may comprise for example waveguide gratings that usediffraction to couple the light beam propagating down toward the chip902 into optical modes that propagate along the waveguides 922 on thechip. As shown, the chip 902 includes a plurality of optical sensors 104each comprising linear waveguides 202 and ring resonators 208. The chip902 additionally includes output couplers 924 that may also comprisewaveguide gratings. These grating couplers 924 similarly use diffractionto couple light propagating in optical modes within the waveguides 202out into free space. Accordingly, light may be injected into the linearwaveguides 202 via an input coupler 922 and extracted therefrom via anoutput coupler 924. As described above, the ring resonators 208 maymodulate this light, for example, shifting a wavelength feature such asthe spectral valley at the resonance wavelength of the ring resonator,depending on whether an object 610 is in proximity of the resonator.

Light from the output couplers 924 is collected by collection optics.The focusing optics 920 can double as the collection optics.Alternatively, separate collection optics may used.

The optical detector 106 (comprising a photodetector 925 in FIG. 9) maybe included in the apparatus 900 to detect the light collected from thechip 902. In some embodiments such as illustrated in FIG. 9, light fromthe output coupler 924 travels to the photodetector 925 via thecollection optics 920, the scanning mirrors 918 as well as abeam-splitter 926 and signal collection optics 928. The scanning mirrors918 can be scanned so as to direct light collected from different outputcouplers 924 and hence different optical sensors 104 at differentlocations on the chip 902.

The apparatus 900 may further comprise an imaging system 930 comprisingimaging optics 932 and an image sensor 934. In some embodiments, thisimage sensor 934 may comprise a single detector that forms an image byrecording the detected signal as the scanning mirrors 918 scan the chip.In some embodiments, this image sensor 934 may comprise a detector arraysuch as a CCD or CMOS detector array.

Light from the chip 902 is collected by the collection optics andpropagates to the imaging system 930 via the scanning mirrors 918, thebeam-splitter 926 (that directs a portion of the light from the outputcoupler 924 to the detector 106), the collimation optics 916, and thesplitter 906 (that also directs light from the laser 904 to the chip).The imaging optics 930 may be used to image the chip 902 and facilitateidentification of which optical sensor 104 is being interrogated at agiven time. Other configurations are possible.

FIG. 10 shows an example of an objective lens 1002 that operates as thefocusing and beam collection optics 920. As illustrated, light isdirected into the input coupling element 922 and returned from theoutput coupling element 924. As illustrated, some embodiments that usegrating couplers 922 and 924, which couple free space light into theon-chip optical elements, eliminate the need for any physical connectionbetween the interrogation apparatus 900 and the chip 902.

Apparatus 900 for interrogating the chip 902 are illustrated in PCTPublication WO 2010/062627 titled Biosensors Based on Optical Probingand Sensing”, which entered the national stage as U.S. application Ser.No. 13/126,164 and which published as U.S. Patent Publication No.2012/0092650 on Apr. 19, 2012, each of which are incorporated herein byreference in their entirety.

The system may vary. For example, instead of using a swept light source,such as a tuneable laser, a broadband light source such as asuper-luminescent diode may be employed.

An example chip 902 is schematically illustrated in FIG. 11. The chip902 includes input and output couplers 922, 924, ring resonators 208 andthe respective waveguides 202 optically coupled thereto. The chip 902further includes flow channels 502 configured to direct flow of solution108 across the optical sensors 104, e.g., the ring resonators 208 andproximal portions of the waveguides 202 optically coupled thereto. Ports1104 for accessing the flow channels 502 are also included to flow thesolution 108 into and out of the flow channels 502.

FIG. 11 shows some 1106 of the optical sensors 104 as having an object610 from the solution 108 coupled to the ring resonators 208. Asdiscussed above, these optical sensors 1106 will have an optical outputindicating this event, such as a shift in the spectral feature at theresonance wavelength of the ring resonator 208.

The chip 902 further includes identification markers 1108 for separatelyidentifying the different optical sensors 104. In some exampleembodiments, identification of the optical sensors 104 is accomplishedusing the imaging system 930 shown in FIG. 9, which images and/orcollects light from the identification markers 1108. In someembodiments, the identification markers 1108 have unique signatures.Additionally, in some embodiments, the identification markers 1108 arediffractive optical elements. In some embodiments, grating couplers 922and 924 may be placed in a distinct pattern that allows the uniqueidentification of each optical sensor 104. Accordingly, in suchembodiments, separate identification markers 1108 need not be included.Other techniques can also be used for identifying the sensors.

One example embodiment of a biosensor chip 902 may be manufactured asfollows. Microring resonator arrays can be fabricated on 8″silicon-on-insulator wafers having, e.g., a top-layer of silicon, fromwhich about 600 individual chips 902 are diced. Each chip 902 hassixty-four ring resonators 208 having 30 μm diameters on a 6×6 mmfootprint. Next to each ring resonator 208 is a linear waveguide 202that has an input diffraction grating coupler 922 and an outputdiffractive grating coupler 924 at either end, allowing the opticalcavity spectrum of each ring resonator 208 to be determinedindependently.

In various embodiments, the surface of each chip 902 is uniformly coatedwith a commercially-available perfluoro (alkenyl vinyl ether) copolymercladding material with windows 404 opened over selected individualsensor elements via photolithography and reactive ion etching. Thiscladding material can serve three purposes: 1) to confine biomoleculeattachment to the active sensing areas of the chip 902, 2) to reduce thenon-specific binding of biomolecules across the surface of the entirechip 902, which might otherwise deplete low abundance targets, and 3) toocclude some ring resonators 208 (those not revealed in the etchingstep) such that these resonators are not exposed to the solution 108,enabling these resonators to be used as controls, for example, forthermal drift.

Sensitivity metrics may be used to compare different types of opticalbiosensors. For example, using saline solution standards the bulkrefractive index sensitivity of an embodiment of this platform wasmeasured to be 7.6×10⁻⁷ refractive index units (RIUs). Using acontrollable polyelectrolyte multilayer growth scheme, the 1/eevanescent field decay length for one embodiment of a high indexcontrast ring resonators 208 was determined to be 63 nm. Additionaldiscussion can be found in (a) Iqbal, M; Gleeson, M A; Spaugh, B; Tybor,F; Gunn, W G; Hochberg, M; Baehr-Jones, T; Bailey, R C; Gunn, L C,Label-Free Biosensor Arrays based on Silicon Ring Resonators andHigh-Speed Optical Scanning Instrumentation. IEEE J. Sel. Top. QuantumElectron 2010, 16, 654-661 as well as Luchansky, M S; Washburn, A L;Martin, T A; Iqbal, M; Gunn, L C; Bailey, R C. Characterization of theevanescent field profile and bound mass sensitivity of a label-freesilicon photonic microring resonator biosensing platform. Biosens.Bioelectron. 2010, doi:10.1016/j.bios.2010.1007.1010, the disclosures ofwhich are hereby incorporated by reference in its entirety. Using amodified radioimmunoassay the surface sensitivity of some sensors 104was determined to be ˜1 pg/mm².

An example apparatus 900 for interrogating the chip 902 having an arrayof biosensors 104 may include laser 904 comprising a tunable, externalcavity diode laser operating with a center wavelength of 1560 nm. A beamfrom the laser 904 is focused onto a single input grating coupler 922and rapidly swept through a suitable spectral bandwidth. The lightcoupled into the input grating coupler 922 is output by thecorresponding output grating coupler 924 and is measured. Resonances aremeasured as wavelengths at which the intensity of light coupled out ofthe output coupler manifest a notch feature. The different ringresonators 208 in the array may be serially interrogated. However, hightuning rate (e.g., kHz) lasers 904 and fast scan mirrors 918 may allowresonance wavelengths and shifts in wavelength to be determined in nearreal time with up to 250 ms temporal resolution. In this embodiment, upto 32 optical sensors 104 can be monitored simultaneously during anexperiment. Any number of the sensors 104 may be left covered by thefluoropolymer cladding and thus may not be exposed to the solution 108and serve as controls for thermal drift. On-chip and real-time driftcompensation can increase sensitivity as temperature dependentrefractive index modulations can obscure biomolecular binding events.On-chip referencing is an effective method of compensating for thissource of noise. Additional discussion is included in Iqbal, M; Gleeson,M A; Spaugh, B; Tybor, F; Gunn, W G; Hochberg, M; Baehr-Jones, T;Bailey, R C; Gunn, L C, Label-Free Biosensor Arrays based on SiliconRing Resonators and High-Speed Optical Scanning Instrumentation. IEEE J.Sel. Top. Quantum Electron 2010, 16, 654-66, the disclosure of which ishereby referenced in its entirety.

Additional details regarding sensors and apparatus for interrogatingsuch sensors are included in U.S. Patent Publication 2011/0045472 titled“Monitoring Enzymatic Process” as well as PCT Publication WO 2010/062627titled “Biosensors Based on Optical Probing and Sensing”, which enteredthe national stage as U.S. application Ser. No. 13/126,164 and whichpublished as U.S. Patent Publication No. 2012/0092650 on Apr. 19, 2012.Each of these documents is incorporated herein by reference in theirentirety.

A wide range of variations, however, are possible. For example, In someembodiments, a ring resonator 208 may be spectrally interrogated bymeans of a broadband light source, such as a superluminescent lightemitting diode (SLED) or erbium amplifier running broadband, thatproduces light having a range of wavelengths all at once, e.g. injectinglight across the input spectrum 210 into waveguide input 204. Likewise,a spectral analyzer (e.g., comprising a spectrometer) may be used tocollect light from waveguide output 206 and analyze output spectrum 212.

Analytes of Interest

The term “analyte” as used herein refers to the substance to be detectedthat may be present in a test sample. Analytes of interest include, butare not limited to polypeptides, nucleic acids, carbohydrates, andantibodies. As used herein with respect to analytes of interest,“nucleic acids” refer to deoxyribonucleic acid (DNA, such as cDNA orgenomic DNA) or ribonucleic acid (RNA). As used herein with respect toanalytes of interest, “polypeptides” refer to peptides of any amino acidlength, which is inclusive of any kind of protein, such as peptidehormones, enzymes and antibodies.

In several embodiments, an analyte of interest is considered abiomarker. The term biomarker commonly refers to a biomolecule usefulfor diagnosing or determining the presence, absence, status, stage, orrisk of developing a particular disease or condition. Generally,biomarkers are differentially present in samples taken from at least twogroups of subjects that differ in health status and can be present at anelevated or decreased level in samples of a first group as compared tosamples of a second group.

In various embodiments, an analyte of interest comprises a ribonucleicacid (RNA). Examples of RNA analytes of interest include, but are notlimited to, messenger RNAs (mRNAs), mRNA splice variants, antisenseRNAs, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs(snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs(siRNAs), tiny non-coding RNAs (tncRNAs), repeat-associated smallinterfering RNAs (rasiRNAs), and microRNAs (miRNAs), and precursor formsof such RNAs.

miRNAs are also known as microRNAs, Mirs, miRs, mirs, and mature miRNAs,and generally refer either to double-stranded intermediate moleculesaround 17 to about 25 nucleotides in length, or to single-strandedmiRNAs, which may comprise a bulged structure upon hybridization with apartially complementary target nucleic acid molecule.

MicroRNAs (miRNAs) are small non-coding RNA molecules encoded in thegenomes of plants and animals. In certain instances, highly conserved,endogenously expressed miRNAs regulate the expression of genes bybinding to the 3′-untranslated regions (3′-UTR) of specific mRNAs. Morethan 1000 different miRNAs have been identified in plants and animalsCertain mature miRNAs appear to originate from long endogenous primarymiRNA transcripts (also known as pri-miRNAs, pri-mirs, pri-miRs orpri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee,et al., EMBO J., 2002, 21(17), 4663-4670). Examples of precursor formsof miRNAs include, but are not limited to, primary miRNA transcripts(also known as pri-pre-miRNAs, pri-mirs, pri-miRs and pri-miRNAs, whichrange from around 70 nucleotides to about 450 nucleotides in length andoften taking the form of a hairpin structure); and pre-miRNAs (alsoknown as pre-mirs, pre-miRs and foldback miRNA precursors, which rangefrom around 50 nucleotides to around 110 nucleotides in length).

Without being bound by theory, the current model of miRNA processinginvolves primary miRNA transcripts being processed by a nuclear enzymein the RNase III family known as Drosha, into approximately 70nucleotide-long pre-miRNAs which are subsequently processed by the DicerRNase into mature miRNAs, approximately 21-25 nucleotides in length. Itis believed that, in processing pri-miRNA into the pre-miRNA, the Droshaenzyme cuts pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′overhang (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, PlantPhysiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee,et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003,425, 415-419). The 3′ two-nucleotide overhang structure, a signature ofRNaseIR cleavage, has been identified as a specificity determinant intargeting and maintaining small RNAs in the RNA interference pathway(Murchison, et al., Curr. Opin. Cell. Biol., 2004, 16, 223-9). Both theprimary RNA transcripts (pri-miRNAs) and foldback miRNA precursors(pre-miRNAs) are believed to be single-stranded RNA molecules with atleast partial double-stranded character, often containing smaller, localinternal hairpin structures.

As used herein, a “sample” or “test sample” can include, but is notlimited to, biological material obtained from an organism or fromcomponents of an organism. The test sample may be of any biologicaltissue or fluid, for example. In some embodiments, the test sample canbe a clinical sample derived from a patient. Examples of test samplesinclude, but are not limited to sputum, cerebrospinal fluid, blood,blood fractions such as serum and plasma, blood cells, tissue, biopsysamples, urine, peritoneal fluid, pleural fluid, amniotic fluid, vaginalswab, skin, lymph fluid, synovial fluid, feces, tears, organs, ortumors. A test sample can also include recombinant cells, cellcomponents, cells grown in vitro, and cell culture constituentsincluding, for example, conditioned medium resulting from the growth ofcells in cell culture medium.

Capture Probes

In several embodiments, capture probes are attached to a surface of anoptical sensor, such as an optical ring resonator. As used herein, a“capture probe” is any molecule that can be used to bind to an analyteof interest.

Without being bound by theory, the resonance wavelengths on the opticalsensor are sensitive to the local refractive index. Biomolecular bindingevents that increase the refractive index at the sensor surface can beobserved as an increase in the resonance wavelength of the opticalsensor. Accordingly, binding of an analyte of interest to a captureprobe attached to a surface of an optical sensor represents a “primary”binding event that can be detected and/or measured in terms of anincrease in the resonance wavelength of the optical sensor of variousembodiments.

Suitable examples of capture probes include, but are not limited to,nucleic acids (e.g. deoxyribonucleic acids and ribonucleic acids),polypeptides (e.g. proteins and enzymes), antibodies, antigens, andlectins. As will be appreciated by one of ordinary skill in the art, anymolecule that can specifically associate with an analyte of interest canbe used as a capture probe. In certain embodiments, the analyte ofinterest and capture probe represent a binding pair, which can includebut is not limited to antibody/antigen (e.g., nucleic acid orpolypeptide), receptor/ligand, polypeptide/nucleic acid, nucleicacid/nucleic acid, enzyme/substrate, carbohydrate/lectin, orpolypeptide/polypeptide. It will also be understood that binding pairsof analytes of interest and capture probes described above can bereversed in several embodiments (e.g. in one embodiment an antibody thatspecifically binds to an antigen can be the analyte of interest and theantigen can be the capture probe, whereas in another embodiment theantibody can be the capture probe and the antigen can be the analyte ofinterest).

The following classes of molecules can be used as capture probes invarious embodiments. It will be understood that such classes ofmolecules are examples only and are not intended to be exhaustive orlimiting.

1. Nucleic Acid Capture Probes

In some embodiments, the capture probe attached to a surface of anoptical sensor can comprise a nucleic acid and is referred to as anucleic acid capture probe. As used herein with respect to captureprobes, “nucleic acid” refers to deoxyribonucleic acid (DNA) orribonucleic acid (RNA) and known analogs, derivatives, or mimeticsthereof. A nucleic acid capture probe can be oligomeric and includeoligonucleotides, oligonucleotides, oligonucleotide analogs,oligonucleotide mimetics and chimeric combinations of these. A nucleicacid capture probe can be single-stranded, double-stranded, circular,branched, or hairpin and can contain structural elements such asinternal or terminal bulges or loops.

In some embodiments, a nucleic acid capture probe can have a length ofat least, or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleobases, or thenucleic acid capture probe can have a length within any range bounded bytwo of the above-mentioned lengths.

In several embodiments, a nucleic acid capture probe and a nucleic acidanalyte of interest bind to form a duplex. Such binding may occurthrough hybridization. As used herein, “hybridization” means the pairingof complementary strands of a nucleic acid capture probe and a nucleicacid analyte of interest. While not limited to a particular mechanism,the most common mechanism of pairing involves hydrogen bonding, whichmay be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of a nucleic acid capture probe and nucleic acid analyte ofinterest.

In some embodiments, a nucleic acid capture probe and nucleic acidmolecule of interest can hybridize under “stringent conditions,” whichrefer to conditions under which a nucleic acid capture probe willhybridize to a nucleic acid molecule of interest, but to a minimalnumber of other sequences. A person of ordinary skill in the art willappreciate that stringent conditions are sequence-dependent and willvary in different circumstances. High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C.

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases of a nucleic acid capture probe andnucleic acid analyte of interest. For example, if a nucleobase at acertain position of a capture probe is capable of hydrogen bonding witha nucleobase at a certain position of a nucleic acid analyte ofinterest, then the position of hydrogen bonding between the captureprobe and the nucleic acid analyte of interest is considered to be acomplementary position. The capture probe and the analyte of interestare complementary to each other when a sufficient number ofcomplementary positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, in some embodiments anucleic acid capture probe and nucleic acid analyte of interest arespecifically hybridizable and complementary, which indicate a sufficientdegree of precise pairing or complementarity over a sufficient number ofnucleobases such that stable and specific binding occurs.

It will be appreciated that the sequence of a nucleic acid capture probeneed not be 100% complementary to that of a nucleic acid analyte ofinterest to be specifically hybridizable. Moreover, a nucleic acidcapture probe may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure, mismatch or hairpin structure). Thenucleic acid capture probes of several embodiments can comprise at least70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%,or at least 92%, or at least 95%, or at least 97%, or at least 98%, orat least 99% sequence complementarity to a region within the nucleicacid sequence of the analyte of interest. The degree of complementarityto be specifically hybridizable can be selected according to well-knownprinciples of hybridization and in accordance with the intendedanalytical procedure.

In several embodiments, a nucleic acid capture probe can comprise one ormore oligonucleotide mimetics. The term “mimetic” includes oligomericnucleic acids wherein the furanose ring or the furanose ring and theinternucleotide linkage are replaced with non-naturally occurringgroups.

In certain embodiments, a nucleic acid capture probe comprises a peptidenucleic acid (PNA) oligonucleotide mimetic (Nielsen et al., Science,1991, 254, 1497-1500). PNAs have favorable hybridization properties,high biological stability and are electrostatically neutral molecules.In PNA oligonucleotide mimetics, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States Patents that teach thepreparation of PNA oligomeric compounds include U.S. Pat. Nos.5,539,082; 5,714,331 and 5,719,262. PNA compounds can be obtainedcommercially from Applied Biosystems (Foster City, Calif., USA).Numerous modifications to the basic PNA backbone are known in the artand can be used in several embodiments.

Another class of oligonucleotide mimetic that can be used for nucleicacid capture probes in several embodiments is linked morpholino units(morpholino nucleic acid) having heterocyclic bases attached to themorpholino ring. A number of linking groups have been reported that linkthe morpholino monomeric units in a morpholino nucleic acid.Morpholino-based oligomeric compounds are non-ionic mimetics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). The morpholino class ofoligomeric compounds has been prepared with a variety of differentlinking groups joining the monomeric subunits.

A further class of oligonucleotide mimetic that can be used for nucleicacid capture probes in several embodiments is cyclohexene nucleic acids(CeNA). In CeNA oligonucleotides, the furanose ring normally present ina DNA or RNA molecule is replaced with a cyclohexenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes.

In several embodiments, a nucleic acid capture probe can comprise alocked nucleic acid (LNA), which can increase the sensitivity andspecificity of conventional oligonucleotides, such as DNAoligonucleotides, for hybridization to short target sequences such asmature miRNAs, stem-loop precursor miRNAs, pre-miRNAs, siRNAs or othernon-coding RNAs as well as miRNA binding sites in their cognate mRNAtargets, mRNAs, mRNA splice variants, RNA-edited mRNAs, antisense RNAsand small nucleolar RNAs (snRNA).

Locked nucleic acid (LNA) capture probes are nucleoside or nucleotideanalogues that include at least one LNA monomer (e.g., an LNA nucleosideor LNA nucleotide). LNA monomers are described in, for example, WO99/14226, U.S. Pat. No. 6,043,060, U.S. Pat. No. 6,268,490, WO 01/07455,WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604. LNAshave bicyclic sugar moieties “in which the 2′-hydroxyl group of theribosyl sugar ring is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage to form the bicyclicsugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs,2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum etal., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The synthesis and preparation of the LNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630).

Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also beenprepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-LNA, a novel conformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In several embodiments, a nucleic acid capture probe can include anon-native, degenerate, or universal base such as inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thioLiracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine, or the like. In some embodiments, a nucleic acid captureprobe can include isocytosine and/or isoguanine in order to reducenon-specific hybridization as generally described in U.S. Pat. No.5,681,702.

In several embodiments, a nucleic acid capture probe can comprise an“aptamer” to bind to a nucleic acid or polypeptide analyte of interest.Aptamers are described in U.S. Pat. Nos. 5,270,163; 5,475,096;5,567,588; 5,595,877; 5,637,459; 5,683,867; and 5,705,337; which areherein incorporated by reference in their entireties. Aptamers can bindto various molecular targets such as small molecules, proteins, andnucleic acids.

2. Polypeptide Capture Probes

In several embodiments, a capture probe attached to a surface of anoptical sensor can comprise a polypeptide, which is inclusive of knownpolypeptide analogs. Examples of polypeptide analogs include moleculesthat comprise a non-naturally occurring amino acid, side chainmodification, backbone modification, N-terminal modification, and/orC-terminal modification known in the art. For example, a polypeptidecapture probe can comprise a D-amino acid, a non-naturally occurringL-amino acid, such as L-(1-naphthyl)-alanine, L-(2-naphthyl)-alanine,L-cyclohexylalanine, and/or L-2-aminoisobutyric acid.

In several embodiments, a polypeptide capture probe can comprise anantigen to which an antibody analyte of interest is capable of binding.In various aspects, a capture probe can comprise a polypeptide antigencapable of binding to an antibody of interest that is a known biomarkerfor a particular disease or condition. It will be appreciated that acapture probe of the systems provided herein can comprise any antigenassociated with any disease or condition for which a subject's antibodyagainst the antigen is considered a biomarker. As a non-limitingexample, a capture probe can comprise a viral antigen capable of bindingto an antibody specific against the viral antigen. Presence of such anantibody, as detected by the systems provided herein, would indicatethat the subject has been infected by the virus and mounted a specificimmune response to it. In certain embodiments, a capture probe cancomprise an auto-antigen associated with an autoimmune disorder or anantigen associated with an allergy, which capture probe is capable ofbinding to an antibody, such as an auto-antibody, of interest. Presenceof such an antibody, as detected by the systems provided herein, wouldindicate that the subject has or is at risk of having the associatedautoimmune disorder or allergy.

3. Lectin Capture Probes

In various embodiments wherein the analyte of interest is acarbohydrate, suitable capture probes can include lectins. Lectins areproteins that bind to saccharides and differ in the types ofcarbohydrate structures they recognize. Several known lectins that canbe used in capture probes of various embodiments include those that havebeen isolated from plants including Conavalia ensiformis, Anguillaanguilla, Triticum vulgaris, Datura stramoniuim, Galanthus nivalis,Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythrinacristagalli, Lens culinaris, Glycine may, Phaseolus vulgaris, Allomyrinadichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, andRicinus communis. Additional lectins that can be used in capture probesof several embodiments include any of the animal, bacterial, or fungallectins known in the art. Several bacterial and fungal lectins haveconsiderably high affinity (micromolar Kd) towards carbohydratescompared to plant or animal lectins.

4. Antibody Capture Probes

In some embodiments, a system for detecting the presence of an analyteof interest includes a capture probe comprising an antibody attached toa surface of an optical sensor. In several embodiments, a capture probecomprising an antibody, referred to herein as an “antibody captureprobe,” is capable of specifically binding a polypeptide analyte ofinterest. As used herein, the term “antibody” includes, but is notlimited to, synthetic antibodies, monoclonal antibodies, recombinantlyproduced antibodies, intrabodies, multispecific antibodies (includingbi-specific antibodies), human antibodies, humanized antibodies,chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (includingbi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

The antibodies of several embodiments provided herein may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may be specific for different epitopes of apolypeptide or may be specific for both a polypeptide as well as for aheterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., PCT publications WO 93/17715; WO 92/08802;WO91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991);U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of which isincorporated herein by reference in its entirety.

Several embodiments are drawn to systems for detecting an analyte ofinterest that is a known biomarker for a particular disease orcondition. In some aspects, the biomarker analyte of interest is amiRNA, overexpressed or underexpressed mRNA, or polypeptide associatedwith a particular disease or condition. Presence of such a biomarker, asdetected by the systems provided herein, would indicate that the subjecthas the disease or condition associated with the biomarker.

Attachment of Capture Probes to Optical Sensor Surface

In several embodiments, the capture probes are attached to a surface ofan optical sensor by a linkage, which may comprise any moiety,functionalization, or modification of the binding surface and/or captureprobes that facilitates the attachment of the capture probes to thesurface of the optical sensor. The linkage between the capture probesand the surface of the optical sensor can comprise one or more chemicalbonds; one or more non-covalent chemical bonds such as Van der Waalsforces, hydrogen bonding, electrostatic interaction, hydrophobicinteraction, or hydrophilic interaction; and/or chemical linkers thatprovide such bonds.

In certain embodiments, the optical sensor surface can have a protectiveor passivating layer to reduce or minimize attachment of molecules otherthan the capture probes. For example, the optical sensor surface can beprotected or passivated to reduce attachment of analyte molecules thatcould otherwise cause false a positive signal or loss of signal.Examples of suitable protective or passivating layers include, but arenot limited to polymers, such as polyethylene glycol (PEG); proteinsthat block nonspecific binding, such as serum albumin and casein;surfactants, such as betaines; carrier nucleic acids, such as salmonsperm DNA; and silicon dioxide.

In several embodiments, the capture probes can be attached to a surfaceof the optical sensor through the use of reactive functional groups onthe capture probes and the surface. For example, a capture probe can beattached to a surface of an optical sensor without a linker byderivatizing the surface with a functional group and contacting thederivatized surface with capture probes.

The functional groups can be functional chemical moieties. For example,the surface of the optical sensor can be derivatized such that achemical functional group on the surface can react with a chemicalfunctional group on the capture probe resulting in attachment. Examplesof functional groups include, but are not limited to, amino, hydroxyl,carboxyl, carboxylate, aldehyde, ester, ether (e.g. thio-ether), amide,amine, nitrile, vinyl, sulfide, sulfonyl, siloxanes, phosphoryl, oxo,thiol, or similar chemically reactive functional groups. Additionalmoieties that can be used as functional groups to attach capture probesto a surface of an optical sensor include, but are not limited to,maleimide, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide,nitrilotriacetic acid, activated hydroxyl, haloacetyl (e.g.,bromoacetyl, iodoacetyl), activated carboxyl, hydrazide, epoxy,aziridine, sulfonylchloride, trifluoromethyldiaziridine,pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone,succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone,isothiocyanate, isocyanate, imidoester, fluorobenzene, biotin andavidin.

In several embodiments, a capture probe can be attached to the surfaceof an optical sensor through a linker, which is often referred to as acrosslinker. Any suitable crosslinker known in the art can be used toattach capture probes to a surface of the optical sensor. Non-limitingexamples of crosslinkers suitable for use in several embodiments includealkyl groups (including substituted alkyl groups and alkyl groupscontaining heteroatom moieties), esters, amide, amine, epoxy groups,ethylene glycol, and derivatives. A crosslinker may also comprise asulfone group, forming a sulfonamide. In some embodiments, a sulfhydryllinker can be used, such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference) which can be used to attach cysteine containingpolypeptides to the surface of an optical sensor. An amino group on thecapture probe can be used for attachment to an amino group on thesurface of an optical sensor. For example, bifunctional groups,including homobifunctional and heterobifunctional linkers commerciallyavailable from Pierce Chemical Company, can be used in severalembodiments.

In some embodiments, a capture probe can be attached to a surface of anoptical sensor via a linker by derivatizing the surface with afunctional group, attaching the derivatized surface to one functionalend of a linker, and attaching a capture probe to the other end of thelinker. Methods of attaching the capture probe to the functionalizedsurface of an optical sensor or crosslinker include reactions that formlinkage such as thioether bonds, disulfide bonds, amide bonds, carbamatebonds, urea linkages, ester bonds, carbonate bonds, ether bonds,hydrazone linkages, Schiff-base linkages, and non-covalent linkages suchas ionic or hydrophobic interactions. It will be appreciated that suchreactions will depend on the type of reactive functional groups on theoptical sensor, or linker, and capture probe.

In some embodiments, a surface of an optical sensor can be coated with athin layer of glass, such as silica (SiOx where x=1-2), using a linkingagent such as a substituted silane, e.g., 3-mercaptopropyl-trimethoxysilane to link the optical sensor to the glass. The glass-coated opticalsensor may then be further treated with a linker, e.g., an amine such as3-aminopropyl-trimethoxysilane, which will function to link theglass-coated optical sensor to the capture probe. Examples of suitablelinkers in various embodiments includeN-(3-aminopropyl)3-mercapto-benzamide, 3-aminopropyl-trimethoxysilane,3-mercaptopropyl-trimethoxysilane, 3-maleimidopropyl-trimethoxysilane,and 3-hydrazidopropyl-trimethoxysilane.

In some embodiments, the capture probe to attach to a surface of anoptical sensor is a nucleic acid capture probe. Any known chemicallyreactive functional group for nucleic acid attachment to a surface canbe used including, but not limited to, aldehyde, epoxy, hydrazide, vinylsulfone, succinimidyl ester, carbodiimide, maleimide, dithio,iodoacetyl, isocyanate, isothiocyanate, aziridine.

In certain embodiments, a nucleic acid capture probe can be attached toa surface of an optical sensor with the S-4FB crosslinker commerciallyavailable from Solulink. The S-4FB linker reacts with primary amines onbiomolecules and converts them to 4-formylbenzamide (4FB) linkermolecules. 4FB-modified molecules form stable hydrazone bonds whenreacted with a(3-N-((6-(N′-Isopropylidene-hydrazino)-nicotinamide)propyltriethyoxysilane)(HyNicSilane, Solulink) modified optical sensor surface.

Density of Capture Probes on an Optical Sensor Surface

In several embodiments, a surface of an optical sensor can have aplurality of the same or different capture probes attached thereto. Thedynamic range of analyte detection can be tuned over several orders ofmagnitude by varying the surface density of the capture probes on thesurface. In such embodiments, the plurality of capture probes canincrease scalability and allow for multiplex analyte detection. In someaspects, a plurality of the same capture probe provides an ability todetect multiple copies of a given analyte of interest. In other aspects,a plurality of different capture probes are attached to a surface of anoptical sensor, thereby permitting multiplex detection of severaldifferent analytes of interest. In some embodiments for detecting miRNAanalytes of interest, an optical sensor can be functionalized withcapture probes for multiple miRNAs. A sample containing the miRNAs ofinterest can be introduced to such an optical sensor and all of themiRNAs can be detected in parallel.

Capture probe density on a surface of an optical sensor can becontrolled, for example, by adjusting the extent of surfacederivatization with a chemically reactive functional moiety. Forexample, capture probe density can be controlled by varying thestoichiometries of a surface reactive functional group, such assiloxane, in the presence of an inert species. It has been demonstratedthat the density of binding sites on a silicon dioxide surface can becontrolled down to <10-7 of a monolayer. Wayment, J. R.; Harris, J. M.Controlling Binding Site Densities on Glass Surfaces. Anal. Chem.2006,78, 7841-7849.

In several embodiments, the capture probes can be attached to a surfaceof an optical sensor at a density of greater than about 0.001 per squaremicrometer, greater than about 0.01 per square micrometer, greater thanabout 0.1 per square micrometer, greater than about 1 per squaremicrometer, greater than about 10 per square micrometer, greater thanabout 100 per square micrometer, greater than about 1000 per squaremicrometer, greater than about 10,000 per square micrometer, greaterthan about 100,000 per square micrometer, greater than about 1,000,000per square micrometer, greater than about 10,000,000 per squaremicrometer, greater than about 100,000,000 per square micrometer,greater than 1,000,000,000 per square micrometer, greater than10,000,000,000 per square micrometer, greater than 100,000,000,000 persquare micrometer, greater than 1,000,000,000,000 per square micrometeror any number in between any of the aforementioned densities. In severalembodiments, a surface of an optical sensor can have a range of captureprobes spanning from a single capture probe to a number of captureprobes that fully saturates all the available binding sites on thesurface.

Antibodies

Similar to a sandwich assay format in which an antigen is first bound bya substrate-immobilized primary capture agent and then recognized by asecondary capture agent, the systems of several embodiments providedherein comprise a capture probe (analogous to a sandwich assay primarycapture agent) and an antibody (analogous to a sandwich assay secondarycapture agent). It is possible to detect and/or measure binding-inducedshifts in the resonance wavelength of individual binding events with thesystems of various embodiments, including binding of an antibody to theoptical sensor. Without being bound by theory, binding of an antibody tothe optical sensor can induce a change in local refractive index,thereby inducing a detectable and/or measurable shift in the resonancewavelength on the optical sensor.

In several embodiments, a system for detecting and/or measuring ananalyte of interest includes an antibody capable of binding to theanalyte of interest or a complex or duplex formed between a captureprobe attached to a surface of an optical sensor and the analyte ofinterest. It will be understood that in several embodiments the antibodycapable of binding to a complex or duplex formed between a capture probeand analyte of interest can bind to a portion of the analyte of interestthat is not bound to the capture probe in formation of the complex orduplex such that the antibody does not directly bind and/or physicallycontact the capture probe. Thus, the binding of a capture probe/analytecomplex by the antibody can be accomplished by the antibody contactingand binding only the analyte portion of the capture probe/analytecomplex. In various aspects, an antibody can bind to an epitope on ananalyte of interest distinct from the epitope or binding site on theanalyte of interest involved in binding to the capture probe. In someaspects, the antibody capable of binding to a complex or duplex formedbetween a capture probe and analyte of interest binds to the analyte ofinterest without inhibiting or interfering with the binding between theanalyte of interest and the capture probe.

An example of a binding event that increases the refractive index at theoptical sensor surface and can be observed as an increase in theresonance wavelength of the optical sensor is an antibody-analytecomplex binding to a capture probe attached to a surface of an opticalsensor (a “primary” binding event). Yet another detectable and/ormeasurable binding event is an antibody binding to an analyte ofinterest which is already bound to a capture probe attached to a surfaceof an optical sensor (a “secondary” binding event). A further detectableand/or measurable binding event is an antibody binding to a duplex orcomplex formed between an analyte of interest and a capture probeattached to a surface of an optical sensor (a “secondary” bindingevent).

It will be understood by a person of ordinary skill in the art that inseveral aspects, an antibody can bind to the analyte of interest eitherprior to or after binding between the analyte of interest and captureprobe. Thus, in some embodiments a binding-induced shift in theresonance wavelength can be detected and/or measured for (1) anantibody-analyte complex binding to a capture probe attached to asurface on an optical sensor, (2) an antibody binding to the analytealready bound to the capture probe attached to a surface on an opticalsensor, or (3) an antibody binding to the duplex or complex formedbetween the analyte and capture probe attached to a surface on anoptical sensor. It will also be apparent to a person of ordinary skillin the art that in some aspects, an antibody is not capable of bindingto the capture probe alone or analyte of interest alone, but is capableof binding to the complex or duplex formed between the capture probe andanalyte of interest.

Accordingly, certain embodiments drawn to a system for detecting ananalyte of interest includes both (1) a capture probe comprising anantibody attached to a surface of an optical sensor and (2) an antibodycapable of binding to the analyte of interest either prior to or afterbinding between the analyte of interest and capture probe. In additionalembodiments, a system for detecting an analyte of interest includes (1)a capture probe comprising a nucleic acid attached to a surface of anoptical sensor wherein the capture probe is capable of binding to ananalyte of interest, and (2) an antibody that is not capable of bindingto the capture probe alone or analyte of interest alone, but is capableof binding to the complex or duplex formed between the capture probe andanalyte of interest.

In certain embodiments, the system includes an antibody thatspecifically binds to an oligonucleotide duplex, such as a DNA:RNAduplex, DNA:DNA duplex, or RNA:RNA duplex, formed between a captureprobe and analyte of interest, but does not bind to the nucleic acidcapture probe or analyte of interest prior to their binding. As usedherein, the term “duplex” refers to a double-stranded molecule, whichcan be formed by hybridization of single-stranded nucleic acids.

Anti-DNA:RNA antibodies can detect miRNA analytes of interest whilesignificantly reducing assay complexity. Both monoclonal and polyclonalantibodies against RNA:RNA and DNA:RNA homoduplexes have been previouslydeveloped and utilized in hybridization based assays for the detectionof numerous nucleic acid targets such as viral nucleic acids and E. colismall RNA. Casebolt, D. B. and C. B. Stephensen, Journal of ClinicalMicrobiology, 1992. 30(3): p. 608-12; Fliss, I., et al., Appl MicrobiolBiotechnol, 1995. 43(4): p. 717-24; Lafer, E. M., et al., J Biol Chem,1986. 261(14): p. 6438-43; Riley, R. L., D. J. Addis, and R. P. Taylor,J Immunol, 1980. 124(1): p. 1-7; Stollar, B. D., FASEB J, 1994. 8(3): p.337-42 and Stollar, B. D. and A. Rashtchian, Anal Biochem, 1987. 161(2):p. 387-94; which are all incorporated by reference in their entireties.

In particular embodiments, a system for detecting an analyte of interestincludes an antibody that specifically binds to a DNA:RNA duplex. Onenon-limiting example of such an antibody that can be used in severalembodiments is that specifically binds to a DNA:RNA duplex is S9.6, amonoclonal antibody that specifically binds to RNA-DNA hybrids asdescribed in Boguslawski et al., J Immunological Methods, 89 (1986)123-130, which is herein incorporated by reference in its entirety.

In several embodiments, the monoclonal antibody S9.6 is used to detect amiRNA analyte of interest. S9.6 is obtained from the hybridoma mousecell line HB-8730, which exhibits sequence independent high bindingaffinity and specificity to RNA:DNA heteroduplexes. Hu, Z., et al.,Nucl. Acids Res., 2006. 34(7): e.52; Székvölgyi, L., et al., Proceedingsof the National Academy of Sciences, 2007. 104(38): p. 14964-14969; andKinney, J. S., et al., Journal of Clinical Microbiology, 1989. 27(1): p.6-12 and Boguslawski, S. J., et al., Journal of Immunological Methods,1986. 89(1): p. 123-130, which are all incorporated by reference intheir entireties. The HB-8730 mouse hybridoma cell line can be obtainedfrom the American Type Culture Collection (ATCC).

Particles

While systems comprising an antibody configured in a sandwich assayformat can detect and/or measure “primary” or “secondary” bindingevents, several embodiments are drawn to systems comprising a particleadapted to amplify a detectable and/or measurable optical property thatis altered (e.g. resonance wavelength) upon a binding event on anoptical sensor. Such embodiments are based on the present discovery thata “secondary” or “tertiary” binding event of particles to an opticalsensor can increase the sensitivity of detection (i.e. lower thedetection limit) by several-fold. For example, a particle can increasethe sensitivity of detection from approximately the low pM to the highfM range, compared to a “secondary” binding event. In certainembodiments, systems can comprise a particle adapted to provide a“primary” binding event detectable signal. For example, a particle canbe bound to an analyte of interest and a complex formed between them canthen be bound to a capture probe attached to a surface of an opticalsensor.

Several embodiments relate to a system for detecting an analyte ofinterest including a particle attached to an antibody, which is capableof specifically binding to the analyte or a duplex or complex formedbetween the analyte and capture probe, or capable of binding to theantibody. The particle is adapted to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor. In one aspect, a particle can bind to an antibody thatis already bound to an optical sensor, whether via binding to an analytewhich is bound to a capture probe attached to a surface of the opticalsensor or binding to a duplex or complex formed between the analyte ofinterest and a capture probe. Such a binding of the particle in thisfashion can be considered a “tertiary” binding event, while the priorbinding of the antibody to the optical sensor is a “secondary” bindingevent and the binding of the analyte of interest to the capture probe isa “primary” binding event.

In various embodiments, a particle can associated with a molecule (e.g.by conjugation) that has affinity for the analyte of interest. Forexample, and not by limitation, a particle can be associated with asilane molecule having affinity to a polypeptide analyte of interest; aparticle can be associated with a phosphate-containing molecule havingaffinity to a nucleic acid analyte of interest; a particle can beassociated with a salt having affinity to a carbohydrate analyte ofinterest; or a particle can be associated with a organic molecule havingaffinity to a lipid.

It will be understood that in several aspects, a particle can beassociated with a molecule that has affinity for the analyte of interestin the same way that capture probes described above can bind to ananalyte of interest. For example, the analyte of interest and moleculeassociated with a particle can represent a binding pair, which caninclude but is not limited to antibody/antigen (nucleic acid orpolypeptide), receptor/ligand, polypeptide/nucleic acid, nucleicacid/nucleic acid, enzyme/substrate, carbohydrate/lectin, orpolypeptide/polypeptide. It will also be understood that binding pairsof analytes of interest and molecules associated with particlesdescribed above can be reversed in several embodiments. Any of thefunctional groups and linkers described above with respect to attachingcapture probes to an optical sensor surface can be used to conjugateparticles to molecules that have affinity to an analyte of interest. Incertain embodiments, an antibody can be conjugated to a particle, suchas a COOH-functionalized polystyrene bead, via a n-hydroxysuccinimideester (NHS) linkage, a DNA molecule can be conjugated to a particle,such as a streptavidin coated glass microsphere via biotin-streptavidinbinding, a carbohydrate molecule can be conjugated to a particle, suchas a gold nanoparticle, via a thiol linkage, a polypeptide molecule canbe conjugated to a particle, such as a titanium dioxide nanoparticle,via an isocyanate silane linkage, and a polypeptide molecule can beconjugated to a particle, such as a magnetic nanoparticle ormicrosphere, via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Itwill also be understood that in various embodiments a molecule that hasaffinity for the analyte of interest can be associated with a particleby passive absorption.

It will be appreciated that a particle can comprise any material, shape,physical state, and/or size sufficient to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor. Without being bound by theory, in some embodiments aparticle comprises any material, shape, physical state, and/or sizesufficient to increase the refractive index at the sensor surface, whichcan be observed as an increase in the resonance wavelength of theoptical sensor. Any particle that has sufficient mass or other physicalproperty, such as electron density, to increase the refractive index atthe sensor surface can be used. In some embodiments, a particle can beamorphous or spherical, cubic, star-shaped, and the like. The particlesprovided herein can comprise solids, liquids, or gasses. In severalembodiments, a particle can comprise crystalline, polycrystalline,polymer, glass, biopolymer, or a composite of these materials.

In some embodiments, a particle adapted to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor has a dimension along any axis, such as an averagediameter, of at least about 0.1 nanometers (nm), 0.5 nm, 1 nm, 5 nm, 10nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800nm, 900 nm, 1,000 nm, 2,000 nm, 3,000 nm, 4,000 nm, 5,000 nm, greaterthan 5,000 nm, any number in between the aforementioned dimensions, orany range between two of the aforementioned dimensions. In severalembodiments, a particle has a dimension along any axis, such as anaverage diameter, of about 1 nm to 1,000 nm. In several embodiments, aparticle has a dimension along any axis, such as an average diameter, ofabout 50 nm to 200 nm.

In some embodiments, a particle comprises a polypeptide of at least 200Daltons, (Da), 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1kilo Dalton (kDa), 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 50 kDa, 75kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800kDa, 900 kDa, 1,000 kDa, 2,000 kDa, 3,000 kDa, 4,000 kDa, 5,000 kDa,6,000 kDa, 7,000 kDa, 8,000 kDa, 9,000 kDa, 10,000 kDa, greater than10,000 kDa, or any size or range between any two of the aforementionedsizes.

In some embodiments, a particle comprises any known polypeptide commonlyused in molecular biology as recombinant expression or purification tagsincluding, but not limited to histidine (His), maltose binding protein(MBP), FLAG, Trx, myc, streptavidin, biotin, human influenza virushemagluttinin (HA), vesicular stomatitis virus glycoprotein (VSV-G),glycoprotein-D precursor of Herpes simplex virus (HSV), V5, AU1,glutathione-S-transferase (GST), the calmodulin binding domain of thecalmodulin binding protein, Protein A, and Protein G. Non-limitingexamples of specific protocols for selecting, making and using anappropriate tag are described in, e.g., Epitope Tagging, pp. 17.90-17.93(Sambrook and Russell, eds., Molecular Cloning A Laboratory Manual, Vol.3, 3rd ed. 2001), which is herein incorporated by reference in itsentirety.

In several embodiments, a particle comprises a nanoparticle, nanosphere,microcapsule, nanocapsule, microsphere, microparticle, bead, colloid,aggregate, flocculate, insoluble salt, emulsion, crystal, detergent,surfactant, dendrimer, copolymer, block polymer, nucleic acid,carbohydrate, lipid, liposome, or insoluble complex. It is contemplatedthat these types of particles can have any size in the picometer,nanometer, micrometer, or millimeter range along any dimensional axis.As used herein, the term “nanoparticle” refers to any particle having agreatest dimension (e.g., diameter) that is less than about 2500 nm. Insome embodiments, the nanoparticle is a solid or a semi-solid. In someembodiments, the nanoparticle is generally centrosymmetric. In someembodiments, the nanoparticle contains a generally uniform dispersion ofsolid components.

Nanoparticles can have a characteristic dimension of less than about 1micrometer, where the characteristic dimension of a particle is thediameter of a perfect sphere having the same volume as the particle. Forexample, the nanoparticle may have a characteristic dimension that isless than 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 180 nm, 150 nm, 120nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm,or any number in between the aforementioned sizes. In some embodiments,the nanoparticle can have a characteristic dimension of 10 nm, 20 nm, 30nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm,180 nm, 200 nm, 250 nm or 300 nm, or any number in between theaforementioned sizes. In other embodiments, the nanoparticle can have acharacteristic dimension of 10-500 nm, 10-400 nm, 10-300 nm, 10-250 nm,10-200 nm, 10-150 nm, 10-100 nm, 10-75 nm, 10-50 nm, 50-500 nm, 50-400nm, 50-300 nm, 50-200 nm, 50-150 nm, 50-100 nm, 50-75 nm, 100-500 nm,100-400 nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150 nm, 150-500 nm,150-400 nm, 150-300 nm, 150-250 nm, 150-200 nm, 200-500 nm, 200-400 nm,200-300 nm, 200-250 nm, 200-500 nm, 200-400 nm or 200-300 nm.

In various embodiments, a particle comprises one or more materialsincluding, but not limited to, polymers such as polystyrene, siliconerubber, latex, polycarbonate, polyurethanes, polypropylenes,polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene. Additional examples of suitable polymers include, but arenot limited to the following: polyethylene glycol (PEG); poly(lacticacid-co-glycolic acid) (PLGA); copolymers of PLGA and PEG; copolymers ofpoly(lactide-co-glycolide) and PEG; polyglycolic acid (PGA); copolymersof PGA and PEG; poly-L-lactic acid (PLLA); copolymers of PLLA and PEG;poly-D-lactic acid (PDLA); copolymers of PDLA and PEG; poly-D,L-lacticacid (PDLLA); copolymers of PDLLA and PEG; poly(ortho ester); copolymersof poly(ortho ester) and PEG; poly(caprolactone); copolymers ofpoly(caprolactone) and PEG; polylysine; copolymers of polylysine andPEG; polyethylene imine; copolymers of polyethylene imine and PEG;polyhydroxyacids; polyanhydrides; polyhydroxyalkanoates,poly(L-lactide-co-L-lysine); poly(serine ester);poly(4-hydroxy-L-proline ester); poly-α-(4-aminobutyl)-L-glycolic acid;derivatives thereof; combinations thereof; and copolymers thereof.

Further examples of polymeric and non-polymeric materials that can beused in particles of several embodiments include, but are not limitedto, poly(lactide), poly(hydroxybutyrate), poly(beta-amino) esters and/orcopolymers thereof. Alternatively, the particles can comprise othermaterials, including but not limited to, poly(dienes) such aspoly(butadiene) and the like; poly(alkenes) such as polyethylene,polypropylene and the like; poly(acrylics) such as poly(acrylic acid)and the like; poly(methacrylics) such as poly(methyl methacrylate),poly(hydroxyethyl methacrylate), and the like; poly(vinyl ethers);poly(vinyl alcohols); poly(vinyl ketones); poly(vinyl halides) such aspoly(vinyl chloride) and the like; poly(vinyl nitriles), poly(vinylesters) such as poly(vinyl acetate) and the like; poly(vinyl pyridines)such as poly(2-vinyl pyridine), poly(5-methyl-2-vinyl pyridine) and thelike; poly(styrenes); poly(carbonates); poly(esters); poly(orthoesters);poly(esteramides); poly(anhydrides); poly(urethanes); poly(amides);cellulose ethers such as methyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose and the like; cellulose esters such ascellulose acetate, cellulose acetate phthalate, cellulose acetatebutyrate; and polysaccharides. These materials may be used alone, asphysical mixtures (blends), or as copolymers.

In several embodiments, a particle comprises a semiconductornanocrystal. A semiconductor nanocrystal is a nanocrystal of Group II-VIand/or Group III-V semiconductor compounds. Examples of semiconductornanocyrstals include, but are not limited to Group II-VI semiconductorssuch as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe,BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well asmixed compositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof.

In several embodiments, a particle comprises a metal particle, such asan Au, Ag, Pd, Pt, Cu, Ni, Co, Fe (e.g. iron sulfide), Mn, Ru, Rh, Os,or Ir particle. In various embodiments, a particle comprises a metaloxide particle. Examples of suitable metal oxide particles include zincoxide, titanium (di)oxide, iron oxide, silver oxide, copper oxide,aluminum oxide, or silicon (di)oxide particles. In certain embodiments,a particle comprises a magnetic particle, such as a magnetic bead,nanoparticle, microparticle, and the like.

In several embodiments, a particle comprises a liposome. Liposomes areunilamellar or multilamellar vesicles which have a membrane formed froma lipophilic material and an aqueous interior. The aqueous interiorportion contains the composition to be delivered. Phospholipids used forliposome formation include, but are not limited to, naturalphospholipids such as egg yolk lecithin (phosphatidyl choline), soybeanlecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidylethanolamine, diphosphatidyl glycerol. Liposome preparation isdescribed, for example, in U.S. Pat. Nos. 7,208,174, 7,108,863,5,192,549, 6,958,241, and in Ann. Rev. Biophys. Bioeng., 9, 467 (1980),“Liposomes” (Ed. by M. J. Ostro, Marcel Dekker, Inc.) the entirecontents of which are incorporated herein by reference.

When phospholipids and many other amphipathic lipids are dispersedgently in an aqueous medium they swell, hydrate and spontaneously formmultilamellar concentric bilayer vesicles with layers of aqueous mediaseparating the lipid bilayers. These systems commonly are referred to asmultilamellar liposomes or multilamellar vesicles (MLV) and usually havediameters of from 0.2 μm to 5 Sonication of MLV results in the formationof small unilamellar vesicles (SUV) with diameters usually in the rangeof 20 to 100 nm, containing an aqueous solution in the core.Multivesicular liposomes (MVL) differ from multilamellar liposomes inthe random, non-concentric arrangement of chambers within the liposomeAmphipathic lipids can form a variety of structures other than liposomeswhen dispersed in water, depending on the molar ratio of lipid to water,but at low ratios the liposome is the preferred structure.

The physical characteristics of liposomes generally depend on pH andionic strength. They characteristically show low permeability to ionicand polar substances, but at certain temperatures can undergo agel-liquid crystalline phase (or main phase) transition dependent uponthe physical properties of the lipids used in their manufacture whichmarkedly alters their permeability. The phase transition involves achange from a closely packed, ordered structure, known as the gel state,to a loosely packed, less-ordered structure, known as the liquidcrystalline state.

Various types of lipids differing in chain length, saturation, and headgroup have been used in liposomal formulations for years, including theunilamellar, multilamellar, and multivesicular liposomes mentionedabove.

There are at least three types of liposomes. The term “multivesicularliposomes (MVL)” generally refers to man-made, microscopic lipidvesicles comprising lipid membranes enclosing multiple non-concentricaqueous chambers. In contrast, “multilamellar liposomes or vesicles(MLV)” have multiple “onion-skin” concentric membranes, in between whichare shell-like concentric aqueous compartments. Multilamellar liposomesand multivesicular liposomes characteristically have mean diameters inthe micrometer range, usually from 0.5 to 25 μm. The term “unilamellarliposomes or vesicles (ULV)” generally refers to liposomal structureshaving a single aqueous chamber, usually with a mean diameter range fromabout 20 to 500 nm.

Multilamellar and unilamellar liposomes can be made by severalrelatively simple methods. A number of techniques for producing ULV andMLV are described in the art (for example in U.S. Pat. No. 4,522,803 toLenk; U.S. Pat. No. 4,310,506 to Baldeschweiler; U.S. Pat. No. 4,235,871to Papahadjopoulos; U.S. Pat. No. 4,224,179 to Schneider, U.S. Pat. No.4,078,052 to Papahadjopoulos; U.S. Pat. No. 4,394,372 to Taylor U.S.Pat. No. 4,308,166 to Marchetti; U.S. Pat. No. 4,485,054 to Mezei; andU.S. Pat. No. 4,508,703 to Redziniak).

By contrast, production of multivesicular liposomes generally requiresseveral process steps. Briefly, a common method for making MVL is asfollows: The first step is making a “water-in-oil” emulsion bydissolving at least one amphipathic lipid and at least one neutral lipidin one or more volatile organic solvents for the lipid component, addingto the lipid component an immiscible first aqueous component and abiologically active substance to be encapsulated, and optionally adding,to either or both the lipid component and the first aqueous component,an acid or other excipient for modulating the release rate of theencapsulated biologically active substances from the MVL. The mixture isemulsified, and then mixed with a second-immiscible aqueous component toform a second emulsion. The second emulsion is mixed eithermechanically, by ultrasonic energy, nozzle atomization, and the like, orby combinations thereof, to form solvent spherules suspended in thesecond aqueous component. The solvent spherules contain multiple aqueousdroplets with the substance to be encapsulated dissolved in them (seeKim et al., Biochem. Biophys. Acta, 728:339-348, 1983). For acomprehensive review of various methods of ULV and MLV preparation,refer to Szoka, et al. Ann. Rev. Biophys. Bioeng. 9:465-508, 1980.

Making multivesicular liposomes can involve inclusion of at least oneamphipathic lipid and one neutral lipid in the lipid component. Theamphipathic lipids can be zwitterionic, anionic, or cationic lipids.Examples of zwitterionic amphipathic lipids are phosphatidylcholines,phosphatidylethanolamines, sphingomyelins etc. Examples of anionicamphipathic lipids are phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, phosphatidic acids, etc. Examples of cationicamphipathic lipids are diacyl trimethylammoniumpropane and ethylphosphatidylcholine. Examples of neutral lipids include diglycerides,such as diolein, dipalmitolein, and mixed caprylin-caprin diglycerides;triglycerides, such as triolein, tripalmitolein, trilinolein,tricaprylin, and trilaurin; vegetable oils, such as soybean oil; animalfats, such as lard and beef fat; squalene; tocopherol; and combinationsthereof. Additionally, cholesterol or plant sterols can be used inmaking multivesicular liposomes.

The liposomes may be made from natural and synthetic phospholipids,glycolipids, and other lipids and lipid congeners; cholesterol,cholesterol derivatives and other cholesterol congeners; charged specieswhich impart a net charge to the membrane; reactive species which canreact after liposome formation to link additional molecules to theliposome membrane; and other lipid soluble compounds which have chemicalor biological activity.

In various embodiments, liposomes can be composed of phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions can be formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes can be formedfrom dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomalcomposition can be formed from phosphatidylcholine (PC) such as, forexample, soybean PC, and egg PC. Another type can be formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of phospholipids suitable for use in several embodimentsinclude but are not limited to DOPC orDC18:1PC=1,2-dioleoyl-sn-glycero-3-phosphocholine; DLPC orDC12:0PC=1,2-dilauroyl-sn-glycero-3-phosphocholine; DMPC orDC14:0PC=1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC orDC16:0PC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC orDC18:0PC=1,2-distearoyl-sn-glycero-3-phosphocholine; DAPC orDC20:0PC=1,2-diarachidoyl-sn-glycero-3-phosphocholine; DBPC orDC22:0PC=1,2-dibehenoyl-sn-glycero-3-phosphocholine;DC16:1PC=1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine;DC20:1PC=1,2-dieicosenoyl-sn-glycero-3-phosphocholine DC22:1PC=1,2-dierucoyl-sn-glycero-3-phosphocholine; DPPG=1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol;DOPG=1,2-dioleoyl-sn-glycero-3-phosphoglycerol.

Furthermore, liposomes of various embodiments can be of various sizes.For example, the average diameter of a liposome in various embodimentscan be about 300 nm, about 295 nm, about 290 nm, about 285 nm, about 280nm, about 275 nm, about 270 nm, about 265 nm, about 260 nm, about 255nm, about 250 nm, about 245 nm, about 240 nm, about 235 nm, about 230nm, about 225 nm, about 220 nm, about 215 nm, about 210 nm, about 205nm, about 200 nm, about 195 nm, about 190 nm, about 185 nm, about 180nm, about 175 nm, about 170 nm, about 165 nm, about 160 nm, about 155nm, about 150 nm, about 145 nm, about 140 nm, about 135 nm, about 130nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm,about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about50 nm, about 45 nm, about 40 nm, about 35 nm, about 30 nm, about 25 nm,about 20 nm, about 15 nm, about 10 nm, or about 5 nm. In certainembodiments, a liposome has a diameter of about 50 nm to 200 nm.

In several embodiments, a particle comprises a surfactant. Surfactantsfind wide application in formulations such as emulsions (includingmicroemulsions) and liposomes. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group (also known as the ‘head’)provides the most useful means for categorizing the differentsurfactants used in formulations (Rieger, in “Pharmaceutical DosageForms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. Popular members of the anionicsurfactant class are the alkyl sulfates and the soaps. Also contemplatedas examples of anionic surfactants that can be used in severalembodiments include stearic acid and sodium behenoyl actylate.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides. The use of surfactants in drugproducts, formulations and in emulsions has been reviewed (Rieger, in“Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y.,1988, p. 285). Preferably such surfactants are nonionic and may be inthe form of silicones or organic nonionic surfactants.

Suitable silicone surfactants include but are not limited topolyorganosiloxane polymers that have amphiphilic properties, forexample contain hydrophilic radicals and lipophilic radicals. Thesesilicone surfactants may be liquids or solids at room temperature.Examples of silicone surfactants that can be used in various embodimentsinclude, but are not limited to: dimethicone copolyols, alkyldimethicone copolyols, and emulsifying silicone elastomers. Emulsifyingsilicone elastomers are elastomers that have one or more hydrophilicgroups such as hydroxyl, oxyethylene, and the like bonded thereto so asto confer hydrophilic properties to the elastomer. Suitable organicnonionic surfactants may include alkoxylated alcohols or ethers formedby the reaction of an alcohol with a polyalkyleneoxide containingrepeating units of alkylene oxide. Preferably, the alcohol is a fattyalcohol having 6 to 30 carbon atoms. Examples of organic nonionicsurfactants that can be used in various embodiments include, but are notlimited to: steareth 2-100, beheneth 5-30, ceteareth 2-100,ceteareth-25, ceteth 1-45, and the like, which are formed bypolyethyleneoxide with the corresponding stearyl/behenyl/cetyl alcohol(wherein the number as used herein designates the number of repeatingunits of ethylene oxide in the polyethyleneoxide). Other alkoxylatedalcohols include esters formed by reaction of polymeric alkylene glycolswith glyceryl fatty acid, such as PEG glyceryl oleates, PEG glycerylstearate; or PEG polyhydroxyalkanotes such as PEG dipolyhydroxystearatewherein the number of repeating ethylene glycol units ranges from 3 to1000. Nonionic surfactants formed by the reaction of a carboxylic acidwith an alkylene oxide or with a polymeric ether are also suitableexamples. Monomeric, homopolymeric, or block copolymeric ethers,alkoxylated sorbitan, alkoxylated sorbitan derivatives can also be usedas nonionic surfactants in various embodiments.

In several embodiments, a particle can be associated with a moleculethat has catalytic activity. Addition of a substrate of the moleculehaving catalytic activity can further amplify a detectable and/ormeasurable optical property that is altered (e.g. resonance wavelength)upon a binding event on an optical sensor. For example, a particle canbe conjugated to horse radish peroxidase (HRP), which can be used toprecipitate a substrate, such as 3,3′-diaminodibenzidine (DAB) onto theoptical sensor, further amplifying a detectable signal (see e.g. FIG.30A).

The particles of various embodiments can comprise a core having any ofthe materials described above or composites thereof, and a surroundingcoat having any of the materials described above or composites thereof.For example, a particle can comprise a magnetic core and a clear coatand/or a coat having a high index dielectric, such as polystyrene. Incertain embodiments, a particle can comprise a core having any of thematerials described above or composites thereof and a coat surroundingthe core having a metal oxide material, such as titanium dioxide, and/ormagnetic material.

Methods of Detecting and/or Measuring the Concentration of Analytes

Several embodiments are drawn to detecting and/or measuring theconcentration of an analyte of interest in a sample using the systemsdescribed above, which can provide for real-time multiplex detection andmeasurement of low abundance biomolecules with high sensitivity andspecificity. It is possible to detect and/or measure binding-inducedshifts in the resonance wavelength of individual binding events inreal-time with the systems of several embodiments. In severalembodiments, “primary,” “secondary,” and “tertiary” binding events canbe applied to an optical sensor and detected and/or measured usingvarious molecule-to-molecule binding assays. In various embodiments,binding events can be detected in real time and/or in multiplex format.In several embodiments, analytes of interest can be detected and/ormeasured at least at the femtomolar (fM) (1×10⁻¹⁵ M) sensitivity range.In various embodiments, an analyte of interest can be present in thesample at least in the femtomolar concentration range or in the picogramper milliliter (pg/mL) range and detected or measured using the systemsdescribed above.

In some embodiments, such binding events detectable in real-time includea “primary” binding event between an analyte of interest (with orwithout a pre-bound particle) and a capture probe (see e.g. FIGS. 30Band 30C), a “secondary” binding event between an antibody (with orwithout a pre-bound particle) and the analyte of interest already boundto the capture probe (see e.g. FIG. 30D), a “secondary” binding eventbetween an antibody (with or without a pre-bound particle) and a duplexor complex formed between the analyte and capture probe, a “secondary”binding event between a particle and the analyte of interest alreadybound to the capture probe (e.g. wherein the capture probe comprises anantigen and the analyte of interest is an antibody against the antigen),or a “tertiary” binding event between a particle and antibody alreadybound to the optical sensor via a “secondary” binding event. Withoutbeing bound by theory, in several embodiments these events inducechanges in local refractive index of the optical sensor, therebyinducing a detectable and/or measurable shift in the resonancewavelength on the optical sensor.

Accordingly, several embodiments are drawn to methods of detecting ananalyte of interest in a sample comprising providing an optical sensor(e.g. optical ring resonator) comprising a capture probe attached to asurface of the optical sensor (e.g. optical ring resonator), wherein thecapture probe is capable of binding to the analyte of interest to form acomplex; applying a sample for which the presence or absence of theanalyte of interest is to be determined to the optical sensor (e.g.optical ring resonator) under conditions in which the analyte ofinterest, when present, and the capture probe bind to form a complex;providing an antibody that specifically binds to the complex or analyte,wherein binding between the antibody and the complex or the analyte,when the analyte is bound to the capture probe, alters an opticalproperty of the optical sensor (e.g. optical ring resonator); anddetermining the presence or absence of the analyte of interest bydetecting the altered optical property of the optical sensor (e.g.optical ring resonator). In some aspects, the concentration of theanalyte of interest in the sample is measured. Detecting and/ormeasuring the concentration of an analyte of interest in a sample can beperformed in real-time and/or in multiplex with other analytes ofinterest or samples.

Certain embodiments relate to methods of detecting an antibody ofinterest, such as an antibody biomarker, including providing an opticalsensor (e.g. optical ring resonator) comprising a capture probe attachedto a surface of the optical sensor (e.g. optical ring resonator),wherein the capture probe comprises an antigen that is capable ofbinding to the antibody of interest to form a complex; applying a samplefor which the presence or absence of the antibody of interest is to bedetermined to the optical sensor (e.g. optical ring resonator) underconditions in which the antibody of interest, when present, and thecapture probe bind to form a complex; providing a detection antibodythat binds to the antibody of interest, wherein binding between thedetection antibody and antibody of interest, when the antibody ofinterest is bound to the capture probe, alters an optical property ofthe optical sensor (e.g. optical ring resonator); and determining thepresence or absence of the antibody of interest by detecting the alteredoptical property of the optical sensor (e.g. optical ring resonator).For example, the antibody of interest can be a human subject'sauto-antibody against an auto-antigen associated with an autoimmunedisorder and the detection antibody can be an anti-human IgG, IgA, orIgM antibody. As another example, the antibody of interest can be ahuman subject's antibody against an antigen associated with an allergyand the detection antibody can be an anti-human IgE antibody. In someaspects, the concentration of the antibody of interest in the sample ismeasured. Detecting and/or measuring the concentration of an antibody ofinterest in a sample can be done in real-time and/or in multiplex withother analytes of interest or samples.

Where the analyte of interest is a nucleic acid molecule, severalembodiments relate to methods of detecting a nucleic acid molecule ofinterest in a sample comprising: providing an optical sensor comprisinga nucleic acid capture probe attached to a surface of the opticalsensor, wherein the capture probe is capable of hybridizing to thenucleic acid molecule of interest to form a duplex; applying a samplefor which the presence or absence of the nucleic acid molecule ofinterest is to be determined to the optical sensor under conditions inwhich the nucleic acid molecule of interest, when present, and thecapture probe sequence-specifically hybridize to form a duplex;providing an antibody that specifically binds a duplex of nucleic acidmolecules, wherein binding between the antibody and the duplex of thecapture probe and nucleic acid molecule of interest alters an opticalproperty of the optical sensor; and determining the presence or absenceof the nucleic acid molecule of interest by detecting the alteredoptical property of the optical sensor. In some aspects, theconcentration of the nucleic acid molecule of interest in the sample ismeasured. Detecting and/or measuring the concentration of a nucleic acidmolecule of interest in a sample can be done in real-time and/or inmultiplex with other analytes of interest or samples.

In some aspects, the nucleic acid molecule of interest is microRNA(miRNA). Despite their roles in cellular processes, miRNAs pose a uniqueset of challenges for their analysis. Short sequence lengths, lowabundance, and high sequence similarity all contribute to make miRNAquantitation difficult using traditional nucleic acid quantitationtechniques such as Northern blotting, reverse transcriptase polymerasechain reaction (RT-PCR), and microarray based detection. Northernblotting, the field standard for miRNA analysis, is a labor and timeintensive process limited by low throughput and large sample volumerequirements. Streit, S., et al., Nat Protoc, 2009. 4(1): p. 37-43. Incontrast, RT-PCR can utilize small sample volumes, but is not wellsuited for quantitative miRNA analysis due to short primers, which oftenreduce the efficiency of the polymerase reaction and introduce signalbias. miRNA analysis is further complicated by the complex nature ofmiRNA-mRNA regulatory networks, as a single miRNA can regulate multiplemRNA targets, or jointly regulate the same mRNA with other miRNAs.

Accordingly, in some embodiments, a miRNA of interest can be detected byproviding an optical sensor comprising a nucleic acid capture probe(e.g. an oligonucleotide comprising DNA and/or LNA) attached to asurface of the optical sensor, wherein the capture probe is capable ofhybridizing to the miRNA of interest to form a duplex; applying a samplefor which the presence or absence of the miRNA of interest is to bedetermined to the optical sensor under conditions in which the miRNA ofinterest, when present, and the capture probe sequence-specificallyhybridize to form a duplex; providing an antibody that specificallybinds a duplex of nucleic acid molecules (e.g. antibody S9.6), whereinbinding between the antibody and the duplex of the capture probe andmiRNA of interest alters an optical property of the optical sensor; anddetermining the presence or absence of the nucleic acid molecule ofinterest by detecting the altered optical property of the opticalsensor. In some aspects, the concentration of the miRNA of interest inthe sample is measured. Detecting and/or measuring the concentration ofa miRNA of interest in a sample can be done in real-time and/or inmultiplex with other analytes of interest or samples.

Amplification of an altered optical property of the optical sensor canbe desirable and accomplished by using a particle described above in adetectable “secondary” or “tertiary” binding event as described above.Use of such particles to amplify an optical detection signal can beuseful in detecting or measuring a low abundance analyte of interest ina sample. In several embodiments, a particle can be used to detectand/or measure an analyte of interest present in a sample at least atthe femtomolar (fM) (1×10⁻¹⁵ M) concentration range or in the picogramper milliliter (pg/mL) range and detected or measured using the systemsdescribed above. In various embodiments, a particle can be used toincrease the dynamic range of detecting and/or measuring an analyte ofinterest present in a sample.

Accordingly, several embodiments are directed to methods of detecting ananalyte of interest in a sample including: providing an optical sensorcomprising a capture probe attached to a surface of the optical sensor,wherein the capture probe is capable of binding to the analyte ofinterest to form a complex; applying a sample for which the presence orabsence of the analyte of interest is to be determined to the opticalsensor, under conditions in which the analyte of interest, when present,and the capture probe bind to form a complex; providing an antibody thatspecifically binds to the complex or analyte, wherein binding betweenthe antibody and the complex or the analyte, when the analyte is boundto the capture probe, alters an optical property of the optical sensor;providing a particle attached to the antibody or a particle capable ofbinding the antibody, wherein the particle amplifies the opticalproperty that is altered; and determining the presence or absence of theanalyte of interest by detecting the altered optical property of theoptical sensor. Use of a particle to amplify a detectable binding eventcan be used in methods to detect any kind of analyte of interestdescribed above, including nucleic acids, polypeptides, and antibodiesin a sample. In some aspects, the concentration of the analyte ofinterest in the sample is measured by methods involving use of aparticle to amplify a detectable binding event. Detecting and/ormeasuring the concentration of an analyte of interest in a sample can bedone in real-time and/or in multiplex with other analytes of interest orsamples by methods involving use of a particle to amplify a detectablebinding event.

In some embodiments, the analyte of interest is an antibody biomarkerfrom a sample obtained from a subject, such as a human patient suspectedof having a disease or condition associated with the antibody biomarker.In one aspect, a sample is applied to an optical sensor to allow anantibody biomarker, if present in the sample, to bind to the captureprobe attached to a surface on the optical sensor. In such an aspect,the capture probe is an antigen to which the antibody biomarker iscapable of binding. Then, either (1) an antibody-specific particle, suchas a bead to which Protein A or Protein G is attached (hereinafterreferred to as a “Protein A bead” or “Protein G bead”), is provided andcan bind to the antibody biomarker bound to the capture probe, (2) adetection antibody (whether or not pre-bound to a particle, includingpre-bound to an antibody specific particle such as a Protein A orProtein G bead) is provided and can bind to the antibody biomarker boundto the capture probe, or (3) the detection antibody is provided firstand then an antibody-specific particle is provided that can bind to thedetection antibody. In any of these aspects, the particle serves toamplify a detectable altered optical property of the optical sensor.

Accordingly, in some embodiments, a particle, such as a Protein A,Protein G, Protein A/G, or Protein L bead, can be provided in a“secondary” binding event to directly bind to the antibody of interest,which already bound in a “primary” binding event to a capture probe.Protein A, G, A/G, and L bind to immunoglobulins. Whereas Protein A, G,and A/G bind to the Fc region of immunoglobulins, Protein L bindsthrough light chain interactions.

In other embodiments, a particle can be pre-bound to the detectionantibody (e.g. an anti-human IgG antibody), and then the resultingcomplex can be provided in a “secondary” binding event to bind to theantibody of interest, which already bound in a “primary” binding eventto a capture probe. For example, a Protein A or Protein G bead can bepre-incubated with a sample to allow binding between the particle andthe antibody biomarker, if present. Then, the particle-antibody complexcan be applied to an optical sensor to allow the complex to bind to thecapture probe. This permits detection and measurement of the antibodybiomarker to the capture probe in real time.

In further embodiments, a particle, such as a Protein A or Protein Gbead, can be provided in a “tertiary” binding event to bind to thedetection antibody, which already bound in a “secondary” binding eventto the antibody of interest, which previously bound in a “primary”binding event to a capture probe.

In additional embodiments, a particle, such as a Protein A or Protein Gbead, can be pre-bound to the antibody of interest, such as a Protein Aor Protein G bead, by incubating a sample from a subject with theparticle under conditions permitting binding. Then, the antibody ofinterest bound to particle can be provided in a “primary” binding eventwith a capture probe.

It will be appreciated that the concentration of the antibody ofinterest in the sample can be measured in the foregoing methods.Detecting and/or measuring the concentration of an antibody of interestin a sample can be done in real-time and/or in multiplex with otheranalytes of interest or samples.

In certain embodiments, the analyte of interest is an antibody from asubject, such as a human, suspected of having an autoimmune disorder. Anautoimmune disorder may include, but is not limited to, diabetesmellitus, transplantation rejection, multiple sclerosis, prematureovarian failure, scleroderm, Sjogren's disease, lupus (e.g. SystemicLupus Erythematosis (SLE)), vitiligo, alopecia (baldness), polyglandularfailure, Grave's disease, hypothyroidism, polymyositis, pemphigus,Crohn's disease, colitis, autoimmune hepatitis, hypopituitarism,myocarditis, Addison's disease, autoimmune skin diseases, uveitis,pernicious anemia, hypoparathyroidism, and/or rheumatoid arthritis.

Accordingly, an auto-antibody analyte of interest can be detected and/ormeasured in a sample in various embodiments using capture probescomprising an autoimmune antigen. Examples of autoimmune antigens thatcan be used as capture probes include, but are not limited to, Jo-1,Smith, SSA, SSB, and Scl-70, RNP, dsDNA, histone/centromere and suchcapture probes can be used to detect and/or measure auto-antibodiesagainst these antigens in a sample. Table 1 provides furthernon-limiting examples of autoimmune antigens associated with variousautoimmune diseases that can be used as capture probes for detectingand/or measure auto-antibody biomarkers.

TABLE 1 Autoimmune Disease Associated Autoantigen(s) Multiple Sclerosismyelin basic protein, proteolipid protein, myelin associatedglycoprotein, cyclic nucleotide phosphodiesterase, myelin- associatedglycoprotein, myelin-associated oligodendrocytic basic protein, myelinoligodendrocyte glycoprotein, alpha-B- crystalin Guillian Barre Syndromeperipheral myelin protein I Diabetes Mellitus tyrosine phosphatase IA2,IA-2β; glutamic acid decarboxylase (65 and 67 kDa forms),carboxypeptidase H, insulin, proinsulin, pre-proinsulin, heat shockproteins, glima 38, islet cell antigen 69 KDa, p52, islet cell glucosetransporter GLUT-2 Rheumatoid Arthritis Immunoglobulin, fibrin,filaggrin, type I, II, III, IV, V, IX, and XI collagens, GP-39, hnRNPsAutoimmune Uveitis protein (IRBP), rhodopsin, recoverin Primary BiliaryCirrhosis pyruvate dehydrogenase complexes (2- oxoacid dehydrogenase)Autoimmune Hepatitis Hepatocyte antigens, cytochrome P450 PemphigusVulgaris Desmoglein-1,-3 Myasthenia Gravis acetylcholine receptorAutoimmune Gastritis H⁺/K⁺ ATPase, intrinsic factor Pernicious Anemiaintrinsic factor Polymyositis histidyl tRNA synthetase, othersynthetases, other nuclear antigens Autoimmune ThyroiditisThyroglobulin, thyroid peroxidase Graves's Disease Thyroid-stimulatinghormone receptor Vitiligo Tyrosinase, tyrosinase-related protein-2Systemic Lupus nuclear antigens: DNA, histones, Celiac DiseaseTransglutaminase

For example, in certain embodiments the antibody biomarker analyte ofinterest is from a subject, such as a human patient suspected of havingan autoimmune disorder. Accordingly, an auto-antibody analyte ofinterest can be detected and/or measured in a sample in variousembodiments using capture probes comprising an autoimmune antigen. Suchauto-antibody biomarkers can be pre-bound to a particle, such as aProtein A or Protein G bead, by incubating a sample from a subject withthe particle under conditions permitting binding. Then, theauto-antibodies bound to particles can be applied to an optical sensorhaving capture probes comprising autoimmune antigens. Examples ofautoimmune antigens that can be used as capture probes include, but arenot limited to, Jo-1, Smith, SSA, SSB, and Scl-70, and those indicatedin Table 1, and such capture probes can be used to detect and/or measureauto-antibodies against these antigens in a sample.

In certain aspects, a sample is applied to an optical sensor to allow anantibody biomarker, if present in the sample, to bind to the captureprobe attached to a surface on the optical sensor. Then, a particle isprovided and can bind to the antibody biomarker bound to the captureprobe. In further aspects, a sample is applied to an optical sensor toallow an antibody biomarker, if present in the sample, to bind to thecapture probe attached to a surface on the optical sensor. Then, ananti-human secondary antibody is provided and can bind to the antibodybiomarker bound to the capture probe. Such anti-human secondary antibodycan be pre-bound to a particle, such as a Protein A or Protein G bead.Alternatively, a particle, such as a Protein A or Protein G bead, can beprovided after the anti-human secondary antibody has bound to theantibody biomarker, which itself is bound to the capture probe.

In several embodiments, binding events can be observed by deterministiccounting methods involving multiple steps. For example, 1°antibody-modified microring resonators can be incubated with the testsample for a defined period. Particle-tagged 2° antibodies can then beadded to quickly saturate the bound analyte of interest. In severalembodiments, the number of discrete shifts in an altered opticalproperty induced by binding events, such as resonance wavelength, over adefined time period can be detected or measured. Deterministic countingmethods can lend themselves to quantitation over a broad dynamic range:the initial slope of antigen binding could be monitored for detection athigh concentrations (μm to low-pM), (Washburn, A L; Gunn, L C; Bailey, RC Label-Free Quantitation of a Cancer Biomarker in Complex Media UsingSilicon Photonic Microring Resonators. Anal. Chem. 2009, 81, 9499-9506),followed by the use of a 2° antibody for intermediate concentrations(low-nM to mid-pM), (Luchansky, M S; Bailey, R C Silicon PhotonicMicroring Resonators for Quantitative Cytokine Detection and T-cellSecretion Analysis. Anal. Chem. 2010, 82, 1975-1981), and then a 3°particle could be introduced via a biotin-streptavidin or anti-IgGinteraction to extend to down to trace levels (mid-pM to low-fM orlower).

In various embodiments, binding events can be observed by stochasticrecording of binding events. For example, particle-tagged 2° antibodiescan be introduced directly into the test sample and allowed to associatewith the small amount analyte, expedited by high relative antibodyconcentrations (2° antibody in excess compared to antigen) and 3-Ddiffusion. After an appropriate time, the shifts in resonance wavelengthare recorded. Since the localization of particles at the sensor surfaceis guided by the interaction between the antigen and capture probe(already on the surface), the shifts in resonance wavelength areexpected to be transient with the binding and unbinding events havingcharacteristic average time constants that directly relate back to theinteraction kinetics.

Stochastic recording methods offer an advantage in that the temporalsignature of binding, as opposed to the magnitude of response, is thequantifiable measure. Accordingly, the signal-to-noise ratio can beincreased by simply integrating over a longer time period. Furthermore,given that T_(off) is not correlated to concentration, but rather isimpacted solely by the dissociation rate constant of the interaction, itmay be possible to distinguish between non-specific and specific bindingevents since non-specific interactions will have shorter residence timesthan specific binding events. In several embodiments, single biomoleculedetection can distinguish between non-specific and specific bindingevents. In a traditional “bulk” experiment where the ensemble of manybinding events is measured, non-specific binding is indistinguishablefrom specific antigen-capture agent interactions. In a time domainmeasurement, T_(off) is not correlated to concentration, but rather isimpacted solely by the dissociation rate constant of the interaction.Non-specific binding events will likely dissociate much faster meaningthat individual unbinding events could be grouped into multiple bins bysimple Fourier transform analysis. In this way, the contributions ofnon-specific binding might be simply filtered out as noise. Thus, inseveral embodiments, trace components can be detected or measured inextraordinarily complex media, such as blood where the dynamic range ofprotein concentration varies over 12 orders of magnitude.

Multiplex Optical Systems

The systems of several embodiments described herein can be used inmultiplex formats and/or in real-time. As used herein, “multiplex” canrefer to a plurality of different capture probes on the same surface ofan optical sensor, or can refer to multiple optical sensors, whereineach sensor can comprise one or more of the same or different captureprobes. In the latter sense, multiple optical sensors can be manipulatedtogether temporally or spatially.

In several embodiments, multiple optical sensors can be manipulated in amultiplex format at the same or different times. For example, multipleoptical sensors can be manipulated simultaneously or at different timesin a multiplex platform, such as a chip, with respect to providingreagent(s) for any of the primary, secondary, or tertiary binding eventsdescribed herein. In some aspects, a test sample can be provided tomultiple optical sensors in a multiplex platform simultaneously. Infurther aspects, an antibody that specifically binds to an analyte ofinterest or a duplex/complex formed between an analyte of interest and acapture probe can be provided to multiple optical sensors in a multiplexplatform simultaneously. In additional aspects, a particle describedherein can be provided to multiple optical sensors in a multiplexplatform simultaneously. In certain aspects, a plurality of the sametype of particle, such as a universal particle, can be provided tomultiple optical sensors in a multiplex platform simultaneously.Multiple optical sensors can also be manipulated simultaneously in amultiplex platform, such as a chip, with respect to detecting ormeasuring the analyte of interest in parallel. In various embodiments,several optical sensors can be independently monitored in a multiplexformat. For example, a plurality of optical rings, wherein each opticalring has a distinct detectable optical property, can be queried ormonitored within the same location, such as in a reaction chamber orsite on a chip, by a single waveguide.

In some embodiments, reagent(s) for any of the primary, secondary, ortertiary binding events described herein can be administered atdifferent times to populations of optical sensors in a multiplexplatform, such as a chip. In other words, a reagent can be provided toone population of optical sensors at a first time, and the reagent canbe provided to another population(s) of optical sensors at differenttime(s), wherein each population comprises one or more optical sensors.In various embodiments, the analyte of interest can be detected in onepopulation of optical sensors at one time and in another population(s)of optical sensors at different time(s), wherein each populationcomprises one or more optical sensors.

In various embodiments, multiple optical sensors can be spatiallymanipulated in a multiplex format. In some aspects, reagent(s) for anyof the primary, secondary, or tertiary binding events described hereincan be differentially administered to distinct populations of opticalsensors in a multiplex platform, such as a chip. In other words, areagent can be provided to one population but not another population ofoptical sensors in a multiplex platform, wherein each populationcomprises one or more optical sensors. In various embodiments, theanalyte of interest can be detected or measured in one population butnot in another population of optical sensors, wherein each populationcomprises one or more optical sensors.

The multiplex embodiments described above are particularly advantageousin reducing cross-talk from the individual detection systems in amultiplex platform. For instance, by temporally or spatiallymanipulating distinct populations of optical sensors in a multiplexplatform, the extent of cross-talk from the individual detection systemscan be reduced. As used herein, the term “cross-talk” refers to abinding event that provides undesired signal detected or measured at anygiven optical sensor. Cross-talk includes false positive signals orinterfering signals resulting from non-specific interaction or bindingof reagents from one detection system and another.

For example, in an immunoassay format in which a detection systemcomprises an antibody capture probe or secondary antibody that iscapable of undesirably cross-reacting with antigens that are notanalytes of interest for a given optical sensor, it is possible toreduce cross-talk by temporally or spatially segregating the source ofcross-talk.

In several embodiments, cross-talk can be temporally reduced byproviding reagent(s) for any of the primary, secondary, or tertiarybinding events described herein at different times. For example,multiple test samples can be provided at different times (e.g. staggeredor sequentially), such that a cross-reacting antigen present in sometest samples but not others cannot result in an undesired signal at agiven time. Also, different secondary antibodies can be provided atdifferent times to reduce non-specific binding of a secondary antibody,which is intended for use with one population of optical sensors, to ananalyte of interest associated with a different population of opticalsensors. In various embodiments, cross-talk can be reduced by detectingor measuring an analyte of interest in different populations of opticalsensors at different times.

Alternatively or additionally, cross-talk can be spatially reduced byproviding reagent(s) for any of the primary, secondary, or tertiarybinding events described herein to distinct populations of opticalsensors in a multiplex platform. For instance, samples havingcross-reacting antigens or secondary antibodies capable ofcross-reacting with an antigen that is not an analyte of interest can bekept separated from distinct populations of optical sensors. In variousembodiments, a multiplex platform can include different flowcells orchannels for providing reagents to spatially separate populations ofoptical sensors in order to reduce cross-talk.

The multiplex embodiments described above are particularly suited forreal-time analyte detection, especially in embodiments with reducedcross-talk. Such binding events detectable in real-time include, but arenot limited to, a “primary” binding event between an analyte of interest(with or without a pre-bound particle) and a capture probe, a“secondary” binding event between an antibody (with or without apre-bound particle) and the analyte of interest already bound to thecapture probe, a “secondary” binding event between an antibody (with orwithout a pre-bound particle) and a duplex or complex formed between theanalyte and capture probe, a “secondary” binding event between aparticle and the analyte of interest already bound to the capture probe,and a “tertiary” binding event between a particle and antibody alreadybound to the optical sensor via a “secondary” binding event.

While various embodiments have been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

EXAMPLES

Having generally described embodiments drawn to systems for detecting ananalyte of interest in a sample and methods of using such systems, afurther understanding can be obtained by reference to certain specificexamples which are provided for purposes of illustration only and arenot intended to be limiting.

Example 1—Optical Sensor Detection of miRNA Fabrication of SiliconPhotonic Microring Resonators and Measurement Instrumentation

Sensor chips were fabricated as described in Washburn et al., AnalyticalChemistry, 2009. 81(22): p. 9499-9506 and Bailey, R. C. et al.,Proceedings of SPIE—The International Society for Optical Engineering,2009, which are herein incorporated by reference in their entireties.

Nucleic Acid Sequences

All synthetic nucleic acids were obtained from Integrated DNATechnologies (“IDT”) (Coralville, Iowa). DNA capture probes were HPLCpurified prior to use, while synthetic RNA probes were RNase Free HPLCpurified. Table 2 shows the sequences of nucleic acid capture probesused in this Example. Sequences of Synthetic Nucleic Acids Bases inunderline indicate the substitution of a locked nucleic acid.

TABLE 2 Sequence (5′ to 3′) hsa miR-16 UAGCAGCACGUAAAUAUUGGCG(SEQ ID NO: 1) hsa miR-21 UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 2)hsa miR-24-1 UGGCUCAGUUCAGCAGGAACAG (SEQ ID NO: 3) hsa miR-26aUUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 4) DNA CaptureNH₂ - (CH₂)₁₂ - ATC GTC GTG Probe CATTTATAACCGC (SEQ ID NO: 5)for hsa miR-16 DNA Capture NH₂ - (CH₂)₁₂ - ATCGAATAGTCTGACT ProbeACAACT(SEQ ID NO: 6) for hsa miR-21 DNA CaptureNH₂ - (CH₂)₁₂ - CTGTTCCTGCTGAACT Probe GAGCCA(SEQ ID NO: 7) for hsa miR-24-1 DNA Capture NH₂ - (CH₂)₁₂ - AAGTTCATTAGGTCCT ProbeATCCGA(SEQ ID NO: 8) for hsa miR- 26a 10 mer RNA AAAGGUGCGU(SEQ ID NO: 9) 20 mer RNA AAAGGUGCGUUUAUAGAUCU (SEQ ID NO: 10)40 mer RNA AAAGGUGCGUUUAUAGAUCUAGACUAGGUUGC AGCAACUA(SEQ ID NO: 11)40 mer DNA NH₂ (CH₂)₁₂ Modular TAGTTGCTGCAACCTAGTCTAGATCTATAAACCapture Probe GCACCTTT(SEQ ID NO: 12) 54 mer DNANH₂ - (CH₂)₁₂ - CTGTTCCTGCTGAACT ModularGAGCCAAAAAAAAAAACTGTTCCTGCTGAACT Capture Probe GAGCCA (SEQ ID NO: 13)LNA Capture NH₂ - (CH₂)₁₂ - CT G TTC CT GCT GAA C ProbeTGAGCCA (SEQ ID NO: 14) for hsa miR- 24-1Modification of ssDNA Capture Probes

DNA capture probes were resuspended in PBS, pH 7.4 upon arrival fromIDT. The probes were buffer exchanged with a new PBS, pH 7.4 solutionthree times utilizing a Vivaspin® 500 Spin column (MWCO 5000, Sartorius)at 10,000 rpm for 6 min to remove any residual ammonium acetate thatwould interfere would the subsequent modifications. A solution ofsuccinimidyl-4-formyl benzoate (S-4FB, Solulink) inN,N-dimethylformamide (Fisher) was added in 4-molar excess to the DNAcapture probe, and allowed to react overnight. The DNA solution wasbuffer exchanged three additional times with PBS, pH 6.0 to remove anyunreacted S-4FB.

Chemical and Biochemical Modification of Silicon Photonic MicroringResonator Surfaces

Prior to treatment, sensor chips were cleaned in a fresh solution ofPiranha (3:1 solution of 16 M H2SO4:30% wt H2O2) for 1 min, andsubsequently rinsed with copious amounts of Millipore H2O. Chips weresonicated for 7 min in isopropanol (Branson 2510 Ultrasonic Cleaner),dried with a stream of N2, and stored until further use.

Chips were immersed in a 1 mg/mL solution of(3-N-((6-(N′-Isopropylidene-hydrazino)-nicotinamide)propyltriethyoxysilane)(HyNicSilane, Solulink) for 30 mM, and afterwards sonicated for 7 mM in100% EtOH to remove any physisorbed HyNic Silane. The chips were driedwith a stream of N2, hand-spotted with 15 μL of DNA modified with a4-molar excess of S-4FB, and allowed to incubate overnight in a humiditychamber. Prior to experiments, the chips were sonicated in 8 M urea for7 min to remove any non-covalently bound capture probe.

Addition of Target miRNA to Sensor Surface

Target miRNA solutions were suspended in a high stringency hybridizationbuffer, consisting of 30% Formamide, 4×SSPE, 2.5×Denhardt's solution(USB Corp.), 30 mM EDTA, and 0.2% SDS, in Millipore H2O. The targetmiRNA solution (35 μL) was recirculated across the sensor surface at arate of 24 μL/min for 1 hr utilizing a P625/10K.133 Instech miniatureperistaltic pump. Solution was delivered to the chip surface via amicrofluidic device consisting of a 0.007″ Mylar gasket sandwichedbetween a Teflon cartridge and the sensor chip. Gaskets were laseretched by RMS Laser in various configurations to allow for multiple flowpatterns.

Blocking and Addition of S9.6

Following addition of the target miRNA to the sensor surface, theInstech peristaltic pump was switched to an 11 Plus syringe pump(Harvard Apparatus) operated in withdraw mode. The chips wereimmediately exposed to Starting Block™ (PBS) Blocking Buffer (ThermoScientific) for 30 min at 10 μL/min to block the sensor surface and helpprevent fouling of S9.6 onto the sensor surface. After, PBS pH 7.4 with0.05% TWEEN® (polysorbate) was flowed over the sensor surface at 30μL/min for 7 mM. A 2 μg/mL solution of S9.6 in PBS, pH 7.4 with 0.05%TWEEN® (polysorbate) was flowed over the sensor surface for 40 min at arate of 30 μL/min

Generation and Purification of the S9.6 Antibody

HB-8730, a mouse hybridoma cell line expressing a monoclonal antibodyhighly specific towards DNA:RNA heteroduplexes, was obtained from theAmerican Type Culture Collection (ATCC). The line was cultured and theS9.6 antibody was purified using Protein G and resuspended at aconcentration of 0.94 mg/mL in PBS, pH 7.4. The antibody solution wasaliquoted and stored at −20° C. until use.

Data Analysis

To utilize the S9.6 response for quantitative purposes, the net sensorresponse after 40 min of exposure to a 2 μg/mL solution of S9.6 wasused. Control rings functionalized with a non-complementary DNA captureprobe were employed to monitor non-specific hybridization-adsorption ofthe target miRNA as well as the non-specific binding of the S9.6antibody. Furthermore, the signal from temperature reference rings(rings buried underneath a polymer cladding layer on the chip) wassubtracted from all sensor signals to account for thermal drift.

Calibration data was fit with the logistic function:

f(c)=(A ₁ /A ₂)/1+(c/c ₀))+A ₂

over a concentration range from 10 pM to 40 nM, with the exception ofmiRNA miR-16 (in which the 40 pM and 10 pM points were not obtained).Fitting Parameters used in generating the logistic function for miR-16,miR-21, miR-24-1, and miR-26a are shown in Table 3.

TABLE 3 Ad- Reduced justed A₁ A₂ x₀ p χ² R² miR-16 −4.05391 822.847865.81162 0.76797 2.12313 0.99675 miR-21 −12.11204 678.14618 2.232780.76436 31.47097 0.98835 miR-24- −35.39772 724.61159 1.61375 0.607470.93902 0.99921 1 miR- 9.22087 753.802 3.2261 0.69393 9.34387 0.9611326a

Results

A schematic of the S9.6 assay is shown in FIG. 12A. The microrings wereinitially functionalized with ssDNA capture probes complementary to thetarget miRNAs of interest. A solution containing the miRNA was flowedacross the sensor surface, after which the surface is blocked with aprotein mixture, and subsequently exposed to the S9.6 antibody. Arepresentative response of 3 microrings corresponding to the schematicis shown in FIG. 12B.

An interesting aspect of the S9.6 antibody was the large signalamplification observed upon S9.6 binding to sensor surfaces, especiallyunder nonsaturating conditions. As shown in FIG. 12B, the net shift forthe hybridization-adsorption of a 100 nM solution of miR-24-1 (aconcentration that will saturate binding sites) onto the sensor surfacewas ˜80 pm. The S9.6 response for amplification was ˜520 pm, limited bysteric crowding of the antibody. However this secondary amplificationbecame even more dramatic at nonsaturating miRNA conditions, increasingthe response over 100-fold.

To determine whether a single DNA:RNA heteroduplex could be bound bymultiple S9.6 antibodies, a sensor surface was created with a single40mer ssDNA capture probe, and subsequently exposed it to three separateRNA sequences, a 10mer, 20mer, and 40mer (Table 2). As shown in FIG. 13,the S9.6 binding response increased significantly from the 10mer to20mer target RNA, indicating that additional antibodies are bound to thesurface, despite the approximately same number of DNA:RNAheteroduplexes. While the increase in S9.6 signal between the 10mer and20mer target RNAs was roughly proportional to the target RNA length,this trend did not hold between the 20mer and 40mer. This could be dueto steric hindrance of the antibodies at the sensor surface; that is,the binding of antibodies to the duplexes prohibited additionalantibodies from reaching potential binding sites closer towards thesensor surface. The S9.6 binding epitope appeared to be <10 base pairsin length, a shorter length than reported previously.

To further interrogate the steric dynamics of S9.6 duplex binding, twossDNA capture probes were designed—a 22mer capture probe completelycomplementary towards miR-24-1, and a second 54mer probe containing twobinding regions completely complementary towards miR-24-1, separated byan A10 spacer. Assuming near saturation of the DNA capture sites withtarget miRNA based on the high concentration of miRNA and ionic strengthof the hybridization buffer, twice as many S9.6 binding sites areavailable on the 54mer capture probe than the 22mer. Furthermore, theA10 stretch in the 54mer capture probe prevents complicatinginteractions between the upper and lower binding sites. As evident inFIG. 14, the S9.6 response for the 54mer capture probes was not doublethose of the 22mer despite the doubling of bound target miRNA,indicating a steric hindrance of the S9.6 binding.

To test the specificity of S9.6, two separate sets of sensors werefunctionalized with ssDNA capture probes complementary towards miR-24-1,and exposed to 1 μM solutions of miR-24-1 and the DNA version of thesame sequence to ensure no sequence bias. A representative S9.6 responsefor an DNA:RNA heteroduplex and DNA:DNA homoduplex were compared in FIG.15A. Even with the sensor surface fully saturated with DNA:DNA duplexes,the non-specific binding response of the S9.6 was 28 pm, ˜6% of theheteroduplex signal, indicating an extremely low non-specific response.

To further gauge the binding properties of the antibody, a sensorcontaining ssDNA and single-stranded locked-nucleic acid (LNA) captureprobes, both complementary towards miR-24-1, was created. LNAs aresynthetic oligonucleotides containing a 2′-O, 4′-C-methylene bridgewhich confers added rigidity to the duplex. Spaced periodically in anoligonucleotide, LNAs have been shown to increase the specificity ofcomplementary sequences and raising the Tm values by 3-8° C. pernucleotide. Even though LNAs convert the ssDNA helix into an A-form fromthe native B-form, as seen in FIG. 15B both the DNA:RNA and LNA:RNAheteroduplexes are bound by S9.6.

Example 2

Multiplex Optical Sensor Detection and Measurement of miRNA Levels inTissue Sample

miRNA levels in mouse brain tissue were measured. The microringresonators, capture probes, and S9.6 antibody were prepared as inExample 1. 50 μg of total mouse brain RNA (Clontech) was diluted 1:5with hybridization buffer and recirculated overnight prior toamplification with S9.6. The net sensor response after 40 min exposureto 2 μg/mL S9.6 was calibrated to each miRNA to account for variable Tmvalues and any secondary structure.

To detect several miRNAs in a sample in multiplex, a single chipcontaining ssDNA capture probes towards miR-16, miR-21, miR-24, andmiR-26a was created. The probes demonstrated no discernable cross-talkeven at high concentrations (FIG. 16), due to the sequencenon-complementarity and high stringency of the hybridization buffer.

The relative expression profiles of the four aforementioned miRNAs inmouse total brain RNA were analyzed. Mouse brain RNA was used due to itscommercial availability as well as literature precedent characterizingthe relative expression of some of the aforementioned miRNAs. Three ofthe sequences are established as being overexpressed in the mouse brain,while expression levels for miR-24-1 have not yet been established. An8-point calibration curve for each of the target miRNAs (with theexception of miR-16, which included 6 separate concentrations) wasgenerated using synthetic miRNAs in buffer on separate chips (FIGS. 17and 18). Table 4 summarizes the average net shifts, standard deviations,and number of measurements for each miRNA, at every concentration usedin generating the calibration curves.

TABLE 4 Average Net Shift Standard Deviation Concentration (Δpm) (Δpm) nmiR-16 40 nM 667.8233 11.68674 6 10 nM 511.1485 20.73502 10 2.56 nM  223.4956 36.97746 10 640 pM  126.107 37.4514 12 160 pM  66.1823221.82598 12  0 pM −4.6422 4.676103 12 miR-21 40 nM 600.5059 4.884918 610 nM 552.0066 8.021747 10 2.56 nM   328.4126 23.88331 7 640 pM 95.49972 12.97273 8 160 pM  67.16636 4.670938 7 40 pM 17.8158 1.7194 1210 pM 9.373472 1.87066 6  0 pM −20.9375 1.896485 12 miR-24-1 40 nM618.8836 20.21606 11 10 nM 537.5413 6.39932 10 2.56 nM   403.696 32.579512 640 pM  239.1976 18.63782 12 160 pM  87.22411 18.20515 10 40 pM40.13903 7.246751 10 10 pM 8.668097 11.29013 11  0 pM −35.7432 2.21001611 miR-26a 40 nM 608.6443 19.12271 11 10 nM 569.5448 14.52657 8 2.56nM   285.0542 18.44371 4 640 pM  185.4222 23.101 9 160 pM  141.018521.39422 11 40 pM 88.14865 24.61825 5 10 pM 13.80172 13.57775 10  0 pM1.818311 10.73274 10

The expression of the aforementioned miRNAs was analyzed in total mousebrain RNA, and after calibration and accounting for the 5 fold dilutionin hybridization buffer, original expression levels were determined tobe 3.12 nM, 0.60 nM, 0.56 nM, and 4.87 nM for miR-16, miR-21, miR-24-1,and miR-26a, respectively (FIG. 19). The overexpression of miR-16 andmiR-26a relative to miR-21 was consistent with previous literaturereports. Table 5 summarizes the S9.6 shifts for total mouse brain RNAand derived concentrations.

TABLE 5 Standard Average Net Deviation Concentration Shift (Δpm) (Δpm) n(nM) miR-16 122.1655 36.76069 8 3.1185 miR-21 54.39331 27.2849 8 0.597miR-24-1 89.7634 23.45957 9 0.557 miR-26a 235.10568 55.97535 9 4.8485

An interesting observation throughout the course of these studies wasthe sigmoidal nature of the S9.6 binding that occurred at high targetmiRNA concentrations. Further experiments with various capture probeconcentrations revealed that the shape of the binding curve was, inpart, dependent on the capture probe density, as shown in FIG. 20. Athigh capture probe densities, the binding becomes sigmoidal in nature,while at low densities, the binding curves take on a logarithmic shapethat characteristic of a Langmuir binding isotherm. It appears that athigh capture probe densities or target probe concentrations, the initialS9.6 binding stabilized the DNA:RNA heteroduplex structure, making iteasier for additional antibodies to bind. This collaborative bindingeffect would explain the sigmoidal shape, but does not account for theslow initial binding rate of the antibody. One possible explanationmight be that the DNA:RNA duplexes acted as an anti-fouling surface forthe antibody. Once S9.6 initially bound to the primarily nucleic acidsurface, it disrupted some of the biofouling properties, allowing otherantibodies to bind nearby as well.

Example 3

Signal Amplification with Nanoparticles

Optical sensor signal amplification was achieved using capture agentstagged with either organic or inorganic nanoparticles. Sequentialimmunoassays were performed for purified interleukins 2 and 4 (IL-2 andIL-4) using biotinylated secondary antibodies against both, but thenincluded a further amplification step for IL-2 via a tertiaryrecognition event with streptavidin-coated CdSe quantum dots.

Materials

3-N-((6-(N′-Isopropylidene-hydrazino))nicotinamide)propyltriethyoxysilane(HyNic silane) and succinimidyl 4-formyl benzoate (S-4FB) were purchasedfrom SoluLink (San Diego, Calif.). Monoclonal mouse anti-human IL-2 andIL-4 (capture antibody, material #555051, clone 5344.111)], monoclonalbiotin mouse anti-human IL-2 (detection antibody, catalog #555040, cloneB33-2) and monoclonal biotin mouse anti-human IL-4 (detection antibody,detection antibody, material #555040, clone B33-2)], in phosphatebuffered saline (PBS) containing 0.09% sodium azide, were purchased fromBD Biosciences (San Jose, Calif.). These served as the primary andsecondary antibodies, respectively. Recombinant human IL-2(catalog#14-8029) in PBS (pH 7.2, with 150 mM NaCl and 1.0% BSA) waspurchased from eBioscience (San Diego, Calif.). PBS was reconstituted indeionized water from Dulbecco's phosphate buffered saline packetspurchased from Sigma-Aldrich (St. Louis, Mo.). Aniline was obtained fromAcros Organics (Geel, Belgium). Phorbol 12-myristate 13-acetate (PMA,Product# P 1585) was purchased from Sigma-Aldrich and dissolved indimethyl sulfoxide to 0.5 mg/mL. The lectin phytohemagglutinin (PHA-P)from Phaseolus vulgaris (Product# L 9132) was also purchased fromSigma-Aldrich and dissolved in PBS, pH 7.4 to 0.5 mg/mL. Zeba spinfilter columns were obtained from Pierce (Rockford, Ill.). Cell culturemedia, RPMI 1640 supplemented with 10% fetal bovine serum (FBS) andpenicillin/streptomycin (100 U/mL each), was obtained from the School ofChemical Sciences Cell Media Facility at the University of Illinois atUrbana-Champaign. All other chemicals were obtained from Sigma-Aldrichand used as received.

Qdot® 525 streptavidin conjugates (CdSe core with ZnS coating) werepurchased as a 1.0 μM solution in 50 mM borate buffer, pH=8.3, with 1.0mM Betaine and 0.05% sodium azide from Molecular Probes, Inc. (catalog#: Q10141MP). Prior to the assay, the quantum dots were diluted to 2 nMin 10 mM PBS pH=7.4 with 0.1 mg/mL BSA.

All buffers and dilutions were made with purified water (ELGA PURELABfiltration system; Lane End, UK), and the pH was adjusted with either 1M HCl or 1 M NaOH. Antibody immobilization buffer was 50 mM sodiumacetate and 150 mM sodium chloride adjusted to pH 6.0. Capture antibodyregeneration buffer was 10 mM glycine and 160 mM NaCl adjusted to pH2.2. BSA-PBS buffer used for IL-2 sensor calibration and detection wasmade by dissolving solid bovine serum albumin (BSA) in PBS (pH 7.4) to afinal concentration of 0.1 mg/mL. For blocking, 2% BSA (w/v) in PBS wasused.

Silicon photonic microring resonator array chips and the instrumentationfor microring resonance wavelength determination were designed incollaboration with and built by Genalyte, Inc. (San Diego, Calif.).Briefly, silicon microring substrates (6×6 mm) contain sixty-fourmicrorings that are accessed by linear waveguides terminated with inputand output diffractive grating couplers, allowing independentdetermination of the resonance wavelength for each microring. Up tothirty-two microring sensors are monitored simultaneously, eight ofwhich are used solely to control for thermal drift. The instrumentationemploys computer-controlled mirrors and a tunable, external cavity diodelaser (center frequency 1560 nm) to rapidly scan the chip surface andsequentially interrogate the array of microring resonators, allowingdetermination of resonance wavelength for each independent sensor with˜250 msec time resolution.

Functionalization of Silicon Photonic Microring Resonator Arrays

Prior to functionalizing the microring surfaces, sensor chips werecleaned by a 30-sec immersion in piranha solution (3:1 H2SO4 30% H2O2)followed by rinsing with copious amounts of water and drying in a streamof nitrogen gas. For all subsequent steps, sensor chips were loaded intoa previously described custom cell with microfluidic flow channelsdefined by a Mylar gasket (Washburn, A. L.; Gunn, L. C.; Bailey, R. C.Anal. Chem. 2009, 81, 9499-9506), and flow was controlled via an 11 Plussyringe pump (Harvard Apparatus; Holliston, Mass.) operated in withdrawmode. Flow rates for functionalization and cytokine detection steps wereset to 5 μL/min. The flow rate was set to 30 μL/min for all additionalsteps.

The chip was first exposed to a solution of 1 mg/mL HyNic silane in 95%ethanol and 5% dimethyl formamide (DMF) for 20 minutes to install ahydrazine moiety on the silicon oxide chip surface, followed by rinsingwith 100% ethanol. In a separate reaction vial, the capture antibody wasfunctionalized with an aldehyde moiety by reacting anti-IL-2 (0.5 mg/mL)with a 5-fold molar excess of 0.2 mg/mL S-4FB (dissolved first in DMF to2 mg/mL for storage and diluted in PBS to 0.2 mg/mL) for 2 hrs at roomtemperature. After buffer-exchanging to remove excess S-4FB using Zebaspin filter columns and dilution to 0.1 mg/mL, the antibody-containingsolution was flowed over the chip to allow covalent attachment to thehydrazine-presenting chip surface. Aniline (100 mM) was added to theantibody solution prior to flowing over the chip, serving as a catalystfor hydrazone bond formation that improves biosensor surfacefunctionalization. The previously-described Mylar gasket (Washburn, A.L.; Gunn, L. C.; Bailey, R. C. Anal. Chem. 2009, 81, 9499-9506) allowsfor selective antibody functionalization on 15 rings under fluidiccontrol. After the coupling reaction, a low-pH glycine-basedregeneration buffer rinse removed any non-covalently bound antibody. Afinal blocking step was carried out by exposing the sensor surface to a2% solution (w/v) of BSA in PBS overnight.

Calibration of Sensors and Detection of IL-2 and IL-4

IL-2 and IL-4 calibration standards were prepared by serial dilution ofrecombinant human IL-2 (≥0.1 mg/mL) and IL-4 in BSA-PBS to the followingconcentrations: 50, 25, 10, 4, 1.6, 0.64, 0.26, 0.10, and 0 ng/mL.Blinded unknown samples were prepared independently from similar stocks.All sandwich assays performed on the chip surface were monitored in realtime and involved a 30-min incubation (5 μL/min) in IL-2 standard, IL-4standard, or unknown solution followed by a 15-min read-out with thesecondary detection anti-IL-2 antibody (2 μg/mL, 5 μL/min) or anti-IL-4antibody. A low-pH glycine buffer rinse, which disrupts non-covalentprotein interactions, was used to regenerate the capture anti-IL-2 andanti-IL-4 surface. The chip was blocked with BSA-PBS prior to subsequentIL-2 and IL-4 detection experiments.

Data Processing

The response from the detection antibody binding to captured IL-2 orIL-4 at the surface as a function of IL-2 or IL-4 concentration was usedto calibrate the sensor response for each ring (n=15 independentmeasurements). Prior to quantitation, the shift response of a controlring, which was not functionalized with capture anti-IL-2 antibody oranti-IL-4 antibody but was exposed to the same solution as thefunctionalized rings, was subtracted from each of the functionalizedring signals to account for any non-specific binding, as well astemperature or instrumental drift. The corrected secondary signal after15 minutes of detection antibody incubation was measured as a net shiftfor each IL-2 and IL-4 standard and unknown, with the signal from eachring serving as an independent measure of IL-2 and IL-4 concentration.The average corrected secondary shift was plotted against concentrationto obtain a calibration plot, which was then fit with a quadraticregression for quantitation of unknowns by inverse regression.

Jurkat Cell Culture, Stimulation, and Secretion Profiling

Jurkat T lymphocytes were passaged into fresh media at 106 cell/mL (10mL culture in each of two T25 vented flasks). One flask was immediatelystimulated to secrete IL-2 and IL-4 by adding the mitogens PMA (50ng/mL) and PHA (2 μg/mL) using an established procedure (Gebert, B.;Fischer, W.; Weiss, E.; Hoffmann, R.; Haas, R. Science 2003, 301,1099-1102, Weiss, A.; Wiskocil, R.; Stobo, J. J. Immunol. 1984, 133,123-128, Manger, B.; Hardy, K. J.; Weiss, A.; Stobo, J. D. J. Clin.Invest. 1986, 77, 1501-1506, Sigma-Aldrich Cat# P1585 Datasheet 2002,http://www.sigmaaldrich.com) while the other flask served as anon-stimulated control. Both flasks were immediately returned to thecell culture incubator (37° C., 5% CO2, 70% relative humidity). Aliquots(1 mL) were withdrawn from both the control and stimulated flasks atfour time points: 0, 8, 16, and 24 hours post-stimulation. The cellculture aliquots were centrifuged at 1,000 RPM for 5 min to pellet thecells, and then the supernatant was removed and centrifuged at 10,000RPM for 5 min to pellet any remaining cellular debris. Cell culturealiquots were divided into two identical tubes and stored for less than24 hours at 4° C. for subsequent parallel analysis by both ELISA and themicroring resonator platform.

A sensor chip was selectively functionalized with anti-IL-2 and IL-4capture antibody as described above and calibrated to secondary antibodyresponse with the following IL-2 and IL-4 standards prepared by serialdilution in cell culture media: 50, 20, 8, 3.2, and 1.3 ng/mLImmediately after calibration, aliquots taken at each time point forboth control and stimulated cells were flowed over all rings on the chip(30 min, 5 μL/min) followed by introduction of the detection anti-IL-2and IL-4 (2 μg/mL, 15 min, 5 μL/min).

Once the rings were functionalized with capture anti-IL-2 and IL-4primary antibodies, a 240-min IL-2 and IL-4 sandwich assay wasperformed, as shown in FIG. 20. IL-4 (130 pM) was added to the rings,followed by addition of secondary anti-IL-4 antibody (13 nM). Next, IL-2(130 pM) was added to the rings, followed by addition of secondaryanti-IL-2 antibody (13 nM). Then, streptavidin-labeled quantum dots wereadded. As shown in FIG. 21, the secondary labels allowed detection downto the order of 5 pM, but the addition of the streptavidin-labeledquantum dots provided a large and specific signal, pushing the assaylimit of detection down to the low 100s of fM.

Example 4

Single Binding Event Detection and Signal Amplification with PolystyreneBeads

To perform single binding event detection, the binding ofcommercially-available protein G-coated polystyrene beads to an array ofantibody modified microring resonators was monitored in real-time asshown in FIG. 22A. Protein G is a bacterial protein that recognized theFC region of antibodies with high affinity, thus facilitatinglocalization of beads onto the microring surface. Microring resonatorswere prepared similar to as in Examples 1 and 3.

As shown in FIG. 22A, protein G-coated polystyrene beads induceddiscrete jumps in relative resonance wavelength shift attributable toindividual binding events of single beads or bead aggregates. This datasuggests that single binding events are easily resolvable, with most ofthe stair step responses being >10σ. A similar experiment was carriedout measuring the binding of streptavidin-modified polystyrene beads tobiotinylated microrings. For this experiment, the number of beads boundto each ring was determined via scanning electron microscopy (SEM) (FIG.22B) and plotted versus the net resonance wavelength shift of thecorresponding ring. The SEA image was stitched from four high resolutionimages and allowed enumeration of beads bound to a given microring. Onlybeads directly contacting the ring, and thus safely within theevanescent field, were counted. As shown in the plot of resonancewavelength shifts versus number of bound beads in FIG. 22C, a cleartrend was observed between sensor response and bead number, providingstrong evidence that single bead binding events are being visualized as“quantized” ˜3.5 pm resonance shifts in real time.

Example 5

Protein-labeled latex beads were used to generate a measurable sensorresponse that directly corresponded to an individual binding event.Microrings functionalized with APTES were covalently labeled with biotinusing a commercial reagent (NHS-PEO4-biotin, Pierce) and avidin-coatedlatex beads introduced to the flow channel. As shown in FIG. 23, thereal-time sensor response showed discrete jumps in resonance frequency,predominantly to higher values consistent with the increase in the localrefractive index due to the binding of a large latex bead. The number ofbeads bound to each ring was determined via scanning electron microscopyand plotted versus the resonance response of the corresponding ring. Atrend was observed between sensor response and bead number providingstrong evidence that single bead binding events were being visualized as“quantized” ˜3.5 pm resonance shifts in real time. Individual stochasticbinding events of bead-labeled biomolecules were detected with theoptical micorring resonators.

Example 6

Optical sensors are used for deterministic counting of binding events.1° antibody-modified microring resonators are incubated with the sampleof interest for a defined period. The solution in the flow cell is thenreplaced with buffer containing a high concentration ofnanoparticle-tagged 2° antibodies so that all of the bound targetmolecules are quickly saturated by the nanoparticle-tagged 2°antibodies. During this process, the number of discrete shifts inresonance wavelength over a defined time period is enumerated.

Example 7

Optical sensors are used for stochastic recording of binding events.Nanoparticle-tagged 2° antibodies are introduced directly into thesample and allowed to associate with the small amount analyte insolution, a process that is expedited by high relative antibodyconcentrations (2° antibody in excess compared to antigen) and 3-Ddiffusion. After an appropriate time, this solution is introduced intothe sensing chamber and the shifts in resonance wavelength are recorded.Since the localization of nanoparticles at the sensor surface is guidedby the interaction between the antigen and 1° antibody (already on thesurface), the shifts in resonance wavelength are expected to betransient with the binding and unbinding events having characteristicaverage time constants that directly relate back to the interactionkinetics. For a simple equilibrium the average time in the “bound”state, T_(off), is related to the dissociation rate constant via,τ_(off)=1/k_(off), and the average time between binding events, τ_(on),is related to the association rate constant and analyte concentration,τ_(on)=1/k_(on)[A], as described in Bayley, H; Cremer, P S Stochasticsensors inspired by biology. Nature 2001, 413, 226-230.

Example 8

Succinimidyl 4-formylbenzoate (S-41-13), succinimidyl6-hydrazinonicotinamide acetone hydrazone (S-HyNic),3-N-((6-(N′-Isopropylidene-hydrazino))nicotinamide)propyltriethyoxysilane(HyNic Silane), and antibody-oligonucleotide conjugation kits wereobtained from SoluLink (San Diego, Calif.). Custom DNA oligonucleotideswere synthesized by Integrated DNA Technologies (Coralville, Iowa).Monoclonal mouse anti-human AFP antibody clone B491M (referred to asanti-AFP-B491M) was purchased from Meridian Life Science, Inc. (Saco,Me.). Monoclonal mouse anti-human AFP antibody clone 2127435 (referredto as anti-AFP-435) were obtained from Fitzgerald IndustriesInternational (Concord, Mass.). Streptavidin-coated polystyrene/ironoxide beads with a mean diameter of 114 nm were purchased from Ademtech(Pessac, France).

Zeba spin filter columns and Starting Block were purchased from Pierce(Rockford, Ill.). Vivaspin molecular weight cutoff filters (both 50,000and 5,000 Da MWCO), were from GE Healthcare (Waukesha, Wis.). Phosphatebuffered saline (PBS, 10 mM phosphate ion concentration) wasreconstituted from Dulbecco's phosphate buffered saline packetspurchased from Sigma-Aldrich (St. Louis, Mo.). All other chemicals wereobtained from Sigma-Aldrich and used as received.

Buffers were prepared with purified water (ELGA PURELAB filtrationsystem; Lane End, UK), and the pH was adjusted with either 1 M HCl or 1M NaOH. PBS buffer with 100 mM phosphate (100 mM PBS) was made with 150mM NaCl, 22.5 mM monobasic sodium phosphate, and 77.7 mM dibasic sodiumphosphate and pH-adjusted to either pH 7.4 or pH 6.0. PBS with tween(PBST, 0.05% Tween-20) was made by adding Tween-20 to standard PBSbuffer (Dulbecco's formulation). All solutions were degassed via vacuumsonication before use.

The microring sensor chip for this experiment was first cleaned withpiranha solution (3:1 H₂SO₄:30% H₂O₂) for 30 seconds followed by rinsingwith water and N₂ drying. To introduce reactive functional groups, thechip was immersed in a 1 mg/mL solution of HyNic Silane (20 mg/mL HyNicSilane in DMF stock solution diluted to 1 mg/mL with ethanol) for 30minutes, followed by rinsing with ethanol and then water.

Oligonucleotides were used to attach primary antibody to the surface andallow the beads to bind to the secondary antibodies. The surface boundantibody was attached via strands B and B′ and the secondary antibodywas functionalized with F′ while the streptavidin beads werefunctionalized with biotinylated strand F.

All oligonucleotides were synthesized with a 5′ amino terminal group tofacilitate attachment to either the substrate or antibody, except forStrand F which had a terminal biotin group. Oligonucleotides werefunctionalized with S-4FB according to manufacturer (SoluLink)instructions. Briefly, oligonucleotides were buffer exchanged to 100 mMPBS pH 7.4 and then a 20-fold molar excess of S-4FB in DMF was added.Solutions were allowed to react overnight at room temperature and thenwere buffer exchanged into 100 mM PBS pH 6.0 using 5 kDa MWCO filters.

HyNic-silane-functionalized chips are DNA-functionalized by manuallypulling a 0.5-μL drop of 4FB-modified strand B (at 150 μM) across thesurface with a 2.5-μL pipette tip. For this experiment, 3 sensors ineach channel were functionalized with strand B with the remainingsensors serving as controls. After spotting the DNA, the drops ofsolution were dried on a hot plate (˜70° C.) and incubated in 80%relative humidity (or higher) for 1-2 hours to allow rehydration of theDNA on the surface. The chip was then immersed into S-4FB-modifiedStarting Block. The Starting Block was modified following the sameprocedure as oligonucleotide functionalization but 100 μL of 5 mg/mLS-4FB was added to 1.5 mL Starting Block. The blocking solution wasremoved by rinsing with water, and then additional S-4FB modifiedblocking solution was added to the chip before incubating overnight in ahumidity chamber at 4° C. The sensor chip was then rinsed with water,and immersed in PBST until use.

To create DNA-antibody conjugates, antibodies were first functionalizedwith S-HyNic following the manufacturer's guidelines. Briefly, S-HyNicin DMF was added in 5-fold molar excess to ˜1 mg/mL antibody that hadpreviously been buffer exchanged into 100 mM PBS pH 7.4 with a Zeba spinfilter and reacted for at least two hours at room temperature. Theantibody was then exchanged into 100 mM PBS pH 6.0 and concentratedusing a 50 kDa MWCO filter, which also served to remove residualS-HyNic. The 4FB-modified DNA was then added in 20-fold molar excess tothe HyNic-modified antibody solution and allowed to react overnight at4° C. DNA-antibody conjugates were then purified away from the excessDNA using a Superdex 200 10/300 GL column on an AKTA FPLC, both from GEHealthcare (Waukesha, Wis.). The separation was performed at 4° C. witha PBS isocratic elution. The collected fractions were concentrated with50 kDa MWCO filters to yield purified solutions of DNA-antibodyconjugates. The final conjugate concentration measured ˜100 μg/mL, asdetermined by measuring the differential absorption at 260 versus 280nm, corresponding to the DNA and IgG, respectively, using a NanoDropUV-Vis absorbance system (ThermoFisher Scientific, Wilmington, Del.).The primary antibody was called B′-anti-AFP (B491M) and the secondaryantibody was called F′-anti-AFP-435.

Streptavidin-coated, 100 nm beads were functionalized with strand F byfirst adding 16 uL of biotinylated strand F (˜300 μM) to 50 μL of 5mg/mL beads. Beads were then buffer exchanged to PBST via magneticseparation and resuspension. They were then diluted to 50 μg/mL prior touse.

The fluidic cell used for this experiment consisted of a 4-channelfluidic cell created by a 0.007-inch thick Mylar gasket topped with apolytetrafluoroethylene (PTFE) top to enable attachment to standardfluidic attachments. Solutions were pulled over the chip via 11 Plussyringe pump (from Harvard Apparatus; Holliston, Mass.) operated inwithdraw mode. For this experiment, each channel had B-anti-AFP (B491M)flowed over the chip until ˜100 pm relative shift was observed on allsensors. Then 0.05, 0.5, 5, and 50 ng/mL AFP were flowed across the chipat 30 μL/min, each in a separate fluidic channel, for ˜30 minutes.Following addition of AFP, 1 ug/mL F′-anti-AFP-435 was flowed across thechip for ˜25 minutes. As a final step, 50 μg/mL 100 nm beads(functionalized with biotinylated strand F) were flowed over the surfacefor ˜20 minutes.

Raw microring resonance wavelength data, recorded as a function of time,was corrected for any thermal drift of bulk refractive index shiftsusing on-chip control rings (exposed to solution, but not functionalizedwith DNA). The signal from all of the control rings was averaged andthen subtracted from each of the individual active sensor rings. Resultsare shown in FIG. 24A-F.

Example 9 Multiplex Detection of Auto-Antibody Biomarkers of Auto-ImmuneDisorders

A multiplex chip was produced having silicon optical rings as describedin Washburn et al., Analytical Chemistry, 2009. 81(22): p. 9499-9506 andBailey, R. C. et al., Proceedings of SPIE—The International Society forOptical Engineering, 2009. Each optical ring was spotted with one of 5antigens (Jo-1, Smith, SSA, SSB, and Scl-70), which are respectivelyassociated with auto-immune diseases polymyositis (PM), systemic lupuserythematosis (SLE), Sjogren's Syndrome and SLE, Sjogren's Syndrome andSLE, and Sjogren's Syndrome.

A serially diluted serum sample positive for all 5 antigens was testedon the multiplex chip and on a commercially available ELISA forcomparison. First, the serum sample was flowed over the multiplex chipand auto-antibodies present in the serum were allowed to bind to theantigen capture probes. Subsequently, beads were flowed over themultiplex chip and allowed to bind to the auto-antibodies thatpreviously bound to the antigen capture probes. Binding between thebeads and auto-antibodies was detected and measured. A schematic of theworkflow is shown in FIG. 25. As shown in FIG. 26, excellent correlationwas observed for all analytes in the multiplex chip. The chip requiredonly 2 μL, whereas ELISA required 50 pL sample volume. Real-time bindingwas observed and results were obtained within 15 minutes.

As shown in FIG. 27, the multiplex chip was up to 10-fold more sensitivethan ELISA at detecting the antigens in terms of dilution. A positivesignal was detected at a 10-fold greater dilution with the multiplexchip as compared to ELISA.

Example 10 Cross-Talk Elimination in Multiplex Optical Detection Systems

Control sera that were known to be positive for 1 or 2 auto-antibodieswere tested at high concentrations to check for cross-talk on amultiplex chip having silicon optical rings, each functionalized withone of 5 antigens (Jo-1, Smith, SSA, SSB, and Scl-70). As shown in FIG.28, no cross-talk was observed, as indicated by the observation that nomore than two binding events were detected for each tested serum sampleknown to have 1 or 2 auto-antibodies.

Example 11 Result Reproducibility of Multiplex Optical Detection Systems

A sample known to be positive for all 5 antigens (Jo-1, Smith, SSA, SSB,and Scl-70) was run on a total of 5 chips, each chip as described inExamples 9 and 10. As shown in Table 6, the results observed were highlyreproducible with a coefficient of variation (CV) less than 15%.

TABLE 6 Coefficient of Antigen Result (pm) Standard Deviation Variation(% CV) Jo-1 113 14 12.5% Smith 201 22 10.8% SSA 654 83 12.7% SSB 201 2512.4% Scl-70 331 36 10.8%

Imaging Based Scatter Detection System

As discussed above, sensitivity can be increased by using refractiveindex tags such as particles or beads in conjunction with the opticalsensor 104. In various embodiments, analyte detection involves a bindingevent wherein a ring resonator 208 captures an analyte and a refractiveindex tag adheres to the captured analyte. The presence of therefractive index tag further increases the refractive index of the ringresonator beyond that induced by the presence of the analyte alone. Theresonance wavelength is thereby shifted to a larger extent. Similarly,the dip in the spectral output 212 from the output waveguide 924 asmeasure by the apparatus 900 for interrogating the sensor chip shifts toa greater extent. The result is increased sensitivity in detection.

Another effect of the refractive index tag, such as a particle or bead,is to increase the scatter of light from the waveguide sensor. Thepresence of the bead in proximity to the waveguide sensor 104 (e.g., thering resonator 208 and/or the waveguide 202 optically coupled thereto)may disrupt the confinement of the light propagating in the ringresonator 208 and/or the waveguide 202 optically coupled thereto andcause the light to scatter out of the waveguide. Some light may leakfrom the waveguide structure (e.g., ring resonator 208) even without thepresence of a bead or other object in proximity to the optical sensor,and more light may be emitted from the resonator at wavelengths injectedinto the resonator that are at the resonance wavelength of theresonator. The presence of the bead or other object is in proximity tothe optical sensor will enhance scattering at the resonance wavelengthof the resonator. (Note that the bead or other object is in proximity tothe optical sensor will shift the resonance wavelength as a result ofthe refractive index of the bead or other object). A binding event thatbrings a bead in close proximity to the sensor 104 could thus bedetected by monitoring radiation exiting the ring resonator 208 and/orthe waveguide 202 optically coupled thereto, for example, using a systemthat images the chip 902 such as the imaging system 930 shown in FIG. 9.Multiple sensors 104, e.g., the entire array of biosensors on a chip 902could be monitored simultaneously; instead of sequentially measuringlight coupled out of the different waveguide couplers 924. Additionally,output grating coupler 924 may not be necessary in some embodiments. Inother embodiments, however, radiation exiting the ring resonator 208and/or the waveguide 202 optically coupled thereto, can be monitored byscanning across the chip 902 and interrogating different optical sensors104 using scanning mirrors 918 and signal collection optics 928 such asshown in FIG. 9. This later embodiment is discussed in connection withFIG. 29A.

In particular, FIG. 29A shows an example apparatus 1200 forinterrogating the optical sensors 104 on a chip 1202 by detectingscattering from individual optical resonators 208. The apparatus 1200includes a laser light source 904, such as a tunable laser, fordirecting light onto the chip 1202. Beam shaping optics such ascollimating optics 916, may be included in the first optical path 1204(indicated by solid arrows) between the laser 904 and the chip 1202 toadjust the shape of the beam as desired. The apparatus 1200 furthercomprises one or more scanning mirrors 918 or other optical elementsconfigured to selectively direct the beam to the appropriate location onthe chip 1202.

The chip 1202 includes input couplers 922 configured to couple the beampropagating in free space into the waveguides 202 on the chip. Asdiscussed above, these input couplers 922 may comprise, for example,waveguide gratings that use diffraction to couple the light beampropagating down toward the chip 1202 into optical modes that propagatealong the waveguides 202 on the chip. The scanning mirrors 918 in theapparatus 1200 for interrogating the optical sensors 104 are moved suchthat the light is directed into the input grating coupler 922 of theoptical sensor 104 to be interrogated.

As shown, the chip 1202 includes a plurality of optical sensors 104 eachcomprising linear waveguides 202 and ring resonators 208. Accordingly,light may be injected into the linear waveguides 202 via an inputcoupler 922 and propagated to the ring resonator 208. A binding eventthat brings a bead or other object in close proximity to the sensor 104(e.g., the ring resonator 208 and/or the waveguide 202 optically coupledthereto) may disrupt the confinement of the light propagating in thering resonator 208 and/or the waveguide 202 optically coupled theretoand cause the light to scatter out of the waveguide into free spacealong a path such as 1206 (indicated by dashed arrows) shown in FIG.29A. This light could thus be detected by monitoring radiation exitingthe optical sensor 104 using collection optics and a detector 1234. Asdiscussed above, the scattering is greater for light having a wavelengthcorresponding to the resonance wavelength of the resonator.Additionally, the presence of the bead or other object in proximity tothe optical sensor will shift this resonance.

In the embodiment shown in FIG. 29A, the focusing optics 920 can doubleas the collection optics. Alternatively, separate collection optics mayused.

The apparatus 1200 in FIG. 29A also includes a detector 1234. Thedetector 1234 is disposed to receive light from the collection optics920. In the particular embodiment shown, light from the chip 1202propagates along second optical path 1206 through the collection optics920 and propagates to the detector 1234 via the scanning mirrors 918 andthe beam-splitter 926. Optional additional optics 1232, labeled imagingoptics in FIG. 29A, may also be included as needed to couple the lightto the detector 1234.

In some embodiments, the apparatus 1200 may be used in conjunction withan imaging system 1230 comprising the imaging optics 1232 and the imagesensor 1234. Accordingly, the imaging system 1230 may be incorporatedinto the apparatus 1200 or it may be separate from the apparatus 1200.In some embodiments, the image sensor 1234 may comprise a detector arraysuch as a CCD or CMOS detector array. The imaging system 1230 may beused to image the chip 1202 and facilitate identification of whichoptical sensor 104 is being interrogated at a given time. Imaging of thechip 1202 may be accomplished alternatively with a single detector asopposed to a detector array, as the scanning mirror 918 enables thedetector's field of view to be scanned across the chip.

Accordingly, as the scanning mirror 918 scans the chip 1202, thedetector 1234 can monitor increases in scattered light from the sensors104. For example, if the field of view of the detector 1234 included anoptical sensor 104 having a ring resonator 208 to which a bead is boundsuch that light propagating within that optical sensor 104 and inparticular within that ring resonator 208 will be scattered into freespace, this light can be detected by the detector 1234. As the scanningmirror 918 scans the chip 1202, optical sensors 104 from which light isemitted into free space will be identified and associated with a bindingevent. As described above, identification markers 1108 may be includedon the chip 1202 and can be used to identify the optical sensors 104. Insome embodiments, the imaging system 1230 is used to read theidentification markers 1108. As described above, in some embodiments,input grating couplers 922 may be placed in a distinct pattern thatallows the unique identification of each optical sensor 104.Accordingly, in such embodiments, separate identification markers 1108need not be included on chip 1202. Other techniques can also be used foridentifying the sensors 104.

Another embodiment of an apparatus 1300 for interrogating the opticalsensors 104 on a chip 1202 is schematically illustrated in FIG. 29B. Theapparatus 1300 includes a laser light source 904, scanning mirrors 918(or other optical elements configured to selectively direct the beam tothe appropriate location on the chip 1202), as well as focusing optics920. The scanning mirrors 918 and focusing optics 920 are included in afirst optical path 1304 (indicated by solid arrows) from the lightsource 904 to the chip 1202. The apparatus 1300 may also includebeamshaping optics 916, which may be included along first optical path1304.

In a manner as described in connection with FIG. 29A, light from thelight source 904 is coupled into respective input grating couplers 922of different optical sensors 104. In the case of a binding event whereina bead or other object is in proximity to the optical sensor 104 so asto scatter light from the waveguide structure, light will be emittedinto free space by the optical sensor.

FIG. 29B differs from FIG. 29A in the approach used to detect thislight. An imaging system 1330 comprising imaging optics 1332 that formsan image of the chip 1202 onto a detector array 1334 is used to monitorlight scattered from the optical sensors 104 by beads attached thereto.The imaging optics 1332 may comprise, for example, one or more lenses.In some embodiments, the detector array 1334 may comprise a CCD or CMOSdetector array. As the lens 1332 forms an image of the chip 1202 on thedetector array 1334, light emitted by optical sensors 104 on the chip1202 will be observable by the detector array.

Although FIG. 29B shows light from a ring resonator 208 of an opticalsensor 104 on the chip 1202 propagating through free space along asecond optical path 1306 to the imaging system 1330, it should be notedthat the imaging optics 1332 may form an image of a larger portion ofthe chip (possibly the entire chip or substantial portions thereof) ontothe detector array 1334. The image formed may thus include scatteredlight from a plurality of optical sensors 104. Imaging a larger portionof the chip 1202 may facilitate identification of the particular sensors104 from which light is scattered by the presence of a bead or otherobject.

As described above, identification markers 1108 on the chip 1202 can bealso used to identify the sensors 104. In the embodiment shown in FIG.29B, the identification markers may also be imaged onto the detectorarray 1334 by the imaging lens 1332. However, as described above, insome embodiments, input grating couplers 922 may be placed in a distinctpattern that allows the unique identification of each optical sensor104. Accordingly, in such embodiments, separate identification markers1108 need not be included on chip 1202. Other techniques can also beused for identifying the sensors 104.

Note that unlike the embodiment in FIG. 29A, the light from the opticssensors 104 does not pass back through the scanning mirrors 918 to reachthe detector array 1334. In fact, in some embodiments, the imagingoptics 1332 and detector array 1334 may be situated on the opposite sideof the chip 1202 such that light is directed into grating couplers 922on one side of the chip (e.g., above the chip) and scattered from thewaveguide sensor 104 in many directions such that a portion is collectedfrom a detector array 1334 located on the other side of the chip (belowthe chip). Likewise, the imaging system 1330 may be incorporated intothe apparatus 1300 or it may be separate from the apparatus 1300.

Other variations are also possible. For example, instead of or inaddition to the scanning mirrors 918 (such as shown in FIGS. 29A and/or29B), actuators (e.g. motors such as stepper motors or piezoelectricdevices) may be used to translate the chip 1202. Alternatively, insteadof using scanning mirrors 918 to direct light into the waveguidestructures, the chip 1202 could be illuminated with less focus, e.g.,flood illumination.

Additionally, the optical spectrum of the light emitted from theresonators can be monitored. As discussed above, the bead or otherobject is in proximity to the resonator will enhance scattering at theresonance wavelength of the resonator. However, the bead or other objectin proximity to the optical sensor will shift the resonance wavelengthof the optical resonator as a result of the refractive index of the beador other object. The light emitted from the resonator via scattering maythus have a spectral peak and that peak may be shifted as well asincreased in magnitude with the binding event involving the bead orother object in proximity to the resonator. Monitoring the spectrum ofemitted light may thus provide additional information.

In some embodiments, in addition to detecting the scatter from theoptical sensors, e.g., ring resonators, output from output waveguides924 such as shown in FIG. 9 (e.g., shift in the dip in the opticalspectrum) can be monitored as described above to provide moreinformation.

Still other variations are possible, for example, in some embodiments,the ring resonator 208 is excluded. For example, a particle coupled tothe linear waveguide 202 can cause the light therein to be decoupled andscattered from the waveguide 202.

1. A system for detecting a nucleic acid molecule of interest in asample comprising: an optical sensor; a nucleic acid capture probeattached to a surface of the optical sensor, wherein the capture probeis capable of hybridizing to the nucleic acid molecule of interest toform a duplex; and an antibody capable of specifically binding to theduplex of the capture probe and nucleic acid molecule of interest,wherein said optical sensor has an optical property that is altered whensaid antibody is bound to said duplex such that said optical sensor isconfigured to sense said antibody combined with said duplex.
 2. Thesystem of claim 1, further comprising: a detector capable of detectingthe optical property that is altered.
 3. (canceled)
 4. The system ofclaim 1, wherein the nucleic acid molecule of interest comprisesdeoxyribonucleic acid (DNA).
 5. The system of claim 1, wherein thenucleic acid molecule of interest comprises ribonucleic acid (RNA). 6.The system of claim 4, wherein the capture probe comprises a DNAoligonucleotide.
 7. The system of claim 6, wherein the DNAoligonucleotide is complementary to the nucleic acid of interest.
 8. Thesystem of claim 6, wherein the DNA oligonucleotide comprises a modifiedDNA nucleotide.
 9. The system of claim 8, wherein the modified DNAnucleotide comprises a locked nucleic acid (LNA).
 10. The system ofclaim 8, wherein the modified DNA nucleotide comprises a universal base.11. The system of claim 1, wherein the capture probe comprises an RNAoligonucleotide.
 12. The system of claim 11, wherein the RNAoligonucleotide is complementary to the nucleic acid of interest oranalyte of interest.
 13. The system of claim 11, wherein the RNAoligonucleotide comprises a modified RNA nucleotide.
 14. The system ofclaim 13, wherein the modified RNA nucleotide comprises a locked nucleicacid (LNA).
 15. The system of claim 14, wherein the modified RNAnucleotide comprises a universal base.
 16. The system of claim 1,wherein the antibody binds to a sequence-independent DNA:RNA duplex anddoes not bind to the nucleic acid molecule of interest or analyte ofinterest prior to the formation of the duplex.
 17. The system of claim16, wherein the antibody is S9.6.
 18. (canceled)
 19. (canceled)
 20. Thesystem of claim 1, wherein the capture probe is covalently coupled tothe surface of the optical sensor or optical ring resonator.
 21. Thesystem of claim 1, wherein the optical sensor comprises a waveguidestructure.
 22. The system of claim 21, wherein the optical sensor has anoutput portion configured to output an optical signal.
 23. The system ofclaim 22, wherein the optical output yields different outputs when saidcapture probe binds to the nucleic acid molecule of interest formingsaid duplex and said antibody binds said duplex, and when said antibodydoes not bind to said duplex.
 24. The system of claim 1, wherein theoptical sensor comprises an input and an output portion each comprisingportions of a waveguide.
 25. The system of claim 24, wherein the opticalsensor comprises an input waveguide and an output waveguide havingoptical coupling region therebetween configured to increase coupling ofa wavelength component from said input waveguide to said outputwaveguide when said capture probe binds to the nucleic acid molecule ofinterest forming said duplex and said antibody binds to said duplex. 26.The system of claim 1, wherein said optical sensor is integrated on anintegrated optical chip comprising optical waveguides.
 27. The system ofclaim 1, wherein the optical sensor comprises a resonator.
 28. Thesystem of claim 27, wherein said resonator has a resonant wavelengththat shifts when said capture probe binds to the nucleic acid moleculeof interest forming said duplex and said antibody binds to said duplex.29. The system of claim 27, wherein the optical sensor comprises awaveguide structure.
 30. The system of claim 27, wherein the opticalsensor comprises a ring resonator.
 31. The system of claim 30, whereinsaid ring resonator comprises a waveguide structure.
 32. The system ofclaim 1, wherein the antibody increases the sensitivity of the opticalsensor in detecting the nucleic acid molecule of interest when theantibody binds to the duplex.
 33. The system of claim 1, wherein theantibody amplifies the optical property that is altered when theantibody binds to the duplex.
 34. A method for detecting a nucleic acidmolecule of interest in a sample comprising: providing an optical sensorcomprising a nucleic acid capture probe attached to a surface of theoptical sensor, wherein the capture probe is capable of hybridizing tothe nucleic acid molecule of interest to form a duplex; applying asample for which the presence or absence of the nucleic acid molecule ofinterest is to be determined to the optical sensor under conditions inwhich the nucleic acid molecule of interest, when present, and thecapture probe sequence-specifically hybridize to form a duplex;providing an antibody that specifically binds a duplex of nucleic acidmolecules, wherein binding between the antibody and the duplex of thecapture probe and nucleic acid molecule of interest alters an opticalproperty of the optical sensor; and determining the presence or absenceof the nucleic acid molecule of interest by detecting the alteredoptical property of the optical sensor.
 35. The method of claim 34,wherein the nucleic acid molecule of interest comprises ribonucleic acid(RNA).
 36. The method of claim 34, wherein the optical sensor comprisesa ring resonator.
 37. The method of claim 36, wherein said ringresonator comprises a waveguide structure. 38-87. (canceled)